Unsymmetrical cyanine dimer compounds and their application

ABSTRACT

Embodiments of the present invention provide methods and nucleic acid reporter molecules for the detection of nucleic acid in a sample. The nucleic acid reporter molecule comprises two unsymmetrical cyanine monomer moieties, which may be the same or different, that are covalently attached by a linker comprising at least one aromatic, heteroaromatic, cyclic or heterocyclic moiety comprising 3-20 non-hydrogen atoms selected from the group consisting of O, N, S, P and C. The linker may be rigid, relatively flexible or some degree thereof. The unsymmetrical cyanine monomer moieties comprise a substituted or unsubstituted benzazolium moiety and a substituted or unsubstituted pyridinium or quinolinium moiety that is connected by a methine bridge that is monomethine, trimethine or pentamethine. The linkers form the cyanine dimer compounds by attaching to the pyridinium or quinolinium moiety of the monomer moieties. The present nucleic acid reporter molecules find utility in forming a nucleic acid-reporter molecule complex and detecting the nucleic acid. In particular, present nucleic acid reporter molecules with a rigid linker and monomer moieties with a monomethine bridge find utility in detecting RNA in the presence of DNA.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. Ser. No. 12/099,020, filed Apr.7, 2008, which is a continuation of U.S. Ser. No. 10/911,423, filed Aug.2, 2004 (now abandoned), which claims priority to U.S. Ser. No.60/491,783, filed Jul. 31, 2003, which disclosures are hereinincorporated by reference in their entirety.

FIELD OF THE INVENTION

The present invention relates to unsymmetrical cyanine dimer compoundsthat provide a detectable fluorescent signal when complexed with nucleicacid polymers. The invention has applications in fields such asmolecular biology, cell biology and fluorescence based assays.

BACKGROUND OF THE INVENTION

The detection of nucleic acid is used in a wide variety of assay formatsto obtain both qualitative and quantitative information about thenucleic acid content of a sample. Fluorescent dyes that complex with DNAand in turn produce a detectable signal have increased the sensitivityand quality of information gained from such experiments. However, therestill remains no fluorescent dye that is capable of detecting RNA in thepresence of DNA for easy and direct detection of RNA.

Currently there exists fluorescent hybridization methods for detectionof RNA in the presence of DNA wherein a dye is covalently attached to anucleic acid hybridization probe (Micklefield, et al. Nucleosides,Nucleotides and Nucleic Acids (2001) 20(4-7), 1169, Yamana, et al.Angew. Chem. Int. Ed. (2001) 40(6), 1104 and Yamana, et al. BioconjugateChemistry (2002) 13, 1266). However, this method is syntheticallyinconvenient and requires many steps to accomplish the desired results.The current invention overcomes the restrictions of the old methods byproviding a simple and efficient means of selective hybridization thatrequires less time and less expertise to accomplish the same or betterresults. Herein we report novel dimer compounds that are capable ofselectively detecting RNA and which can also be used for the detectionof DNA.

Dimers that are known for the detection of nucleic acid include dimersof unsymmetrical cyanine dyes, (U.S. Pat. Nos. 5,410,030; 5,582,977;6,664,047 and WO 93106482) ethidium dimers (U.S. Pat. No. 5,314,805),acridine dimers and acridine-ethidium heterodimers (U.S. Pat. No.6,428,667 and Rye, et al. Nucleic Acids Research (1990) 19(2), 327). Thefollowing references describe DNA intercalating fluorescent dimers andtheir physical characteristics: Gaugain et al., Biochemistry (1978)17:5071-5078; Gaugain et al., Biochemistry (1978) 17:5078-5088;Markovits et al., Anal. Biochemistry (1979) 94:259-269; Markovits et al.Biochemistry (1983) 22: 3231-3237; and Markovits et al., Nucl. AcidsRes. (1985) 13:3773-3788. The present dimers that are described hereinare not only different in structure from other dimer compounds, butdiffer in spectral properties, binding affinities, intracellularproperties and binding kinetics.

The present invention overcomes the limitations of the known nucleicacid reporter molecules by providing reporter molecules that are capableof detecting RNA in the presence of DNA. These novel dimer compounds areattached by a linker that contains at least one aromatic,heteroaromatic, cyclic or heterocyclic moiety. The rigidity of thismoiety is one factor that allows for the ability to selectively detectRNA. In addition, the present invention also provides reporter moleculesthat have utility for detecting DNA.

SUMMARY OF THE INVENTION

Embodiments of the present invention provide nucleic acid reportermolecules that are dimers of unsymmetrical cyanine dyes covalentlyattached by a linker that contains at least one aromatic,heteroaromatic, cyclic or heterocyclic moiety. The linker comprises 3-20non-hydrogen atoms selected from the group consisting of O, N, S, P andC. The linkers may be flexible, rigid or some degree thereof. Thepresent nucleic acid reporter molecules find utility in detectingnucleic acid polymers wherein the dimer compounds complex with nucleicacid and provide a detectable signal. These nucleic acid polymers aresingle, double, triple or quadruple stranded DNA or RNA. Typically theDNA is single or double stranded and the RNA is single stranded. In oneaspect of the present invention, the nucleic acid reporter moleculesdetect RNA in the presence of DNA by producing a fluorescent intensitysignal that is greater on RNA than on DNA.

The linkers are typically represented by Formula (I)—(Y)_(r)—(CH₂)_(m)-T_(q)-(CH₂)_(n)-E-(CH₂)_(n)-T_(q)-(CH₂)_(m)—(Y)_(r)—and Formula (II) —(CH₂)_(n)-T_(q)-(CH₂)_(n)-E-(CH₂)_(n)-T_(q)-(CH₂)_(n)—wherein Formula (II) typically represents rigid linkers. According toformula (I) and (II), when present, Y is a linear or branched moietycomprising 1-20 non-hydrogen atoms selected from the group consisting ofC, N, O, P and S; H is a heteroatom; E is an aromatic, heteroaromatic,cyclic or heterocyclic moiety comprising 3-20 non-hydrogen atomsselected from the group consisting of O, N, S, P and C; m is 0-6 and r,n and q are independently 0 or 1. Rigid linkers comprise an aromatic,heteroaromatic, cyclic or heterocyclic moiety that is either directlybonded to the cyanine monomers or contains an additional 1-6non-hydrogen atoms that directly connect the moiety to the cyaninemonomers.

In an exemplary embodiment, the present compounds are according to theformula:

-   -   wherein each R³ is independently hydrogen, unsubstituted C₁-C₆        alkyl, substituted C₁-C₆ alkyl, unsubstituted C₁-C₆ alkoxy,        substituted C₁-C₆ alkoxy, unsubstituted fused benzene,        substituted fused benzene, unsubstituted trifluoromethyl,        substituted trifluoromethyl, unsubstituted halogen, substituted        halogen, unsubstituted reactive group, substituted reactive        group, unsubstituted carrier molecule, substituted carrier        molecule, unsubstituted solid support or substituted solid        support;    -   each R⁴ is independently a unsubstituted C₁-C₆ alkyl,        substituted C₁-C₆ alkyl, unsubstituted reactive group,        substituted reactive group, unsubstituted carrier molecule,        substituted carrier molecule, unsubstituted solid support or        substituted solid support;    -   X is O, S, or CR⁶R⁷ wherein each R⁶ and R⁷ are independently a        unsubstituted C₁-C₆ alkyl, substituted C₁-C₆ alkyl,        unsubstituted reactive group, substituted reactive group,        unsubstituted carrier molecule, substituted carrier molecule,        unsubstituted solid support, substituted solid support or R⁶ and        R⁷ taken together form a 5- or 6-membered saturated ring;    -   j is 0, 1, or 2;    -   linker is a series of stable covalent bonds comprising 1-30        nonhydrogen atoms selected from the group consisting of C, N, O,        S and P; and,    -   K is substituted pyridinium, unsubstituted pyridinium,        substituted quinolinium, or unsubstituted quinolinium.

While unsymmetrical cyanine monomer moieties are well recognized fortheir ability to complex with and detect DNA, the formation of dimercompounds with particular cyanine monomer moieties and certain rigidlinkers according to Formula (II) results in unexpected advantageswherein these reporter molecules are capable of detecting RNA in thepresence of DNA. For these RNA reporter molecules, the cyanine monomerstypically comprise a monomethine bridge and the linker is typicallyselected from the group consisting of:

Thus, in one aspect of the invention, these RNA reporter molecules areintensely fluorescent when associated with RNA and only dimly or not atall fluorescent when associated with DNA. Therefore, the present cyaninedimer compounds comprising a linker according to Formula (I) and (II)are useful for detecting single, double, triple or quadruple strandednucleic acid whereas a subset of these reporter molecules areparticularly useful for selectively detecting RNA in the presence ofDNA. Alternatively, a subset of these reporter molecules, at certainconcentrations, are particularly useful for selectively detectingmitochondrial DNA.

Additional embodiments of the present invention provide kits for thedetection of nucleic acid, wherein the kit comprises any compound of thepresent invention. In a further embodiment, the kits compriseinstructions for the detection of nucleic acid, particularlyinstructions for the detection of intracellular RNA. In yet anotherfurther embodiment, the kits comprises at least one component that is asample preparation reagent, a buffer agent, an organic solvent or anaddition nucleic acid reporter molecule.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Shows the intensity of the fluorescent signal from Compound 6[1-2 μM] (FIG. 1A) and Compound 19 [1-2 μM] (FIG. 1B) bound to RNA orDNA in solution. Both compounds demonstrated a 4- to 8-fold increase insignal intensity when bound to RNA compared to DNA. See, Example 35.

FIG. 2: Shows the intensity of the fluorescent signal from Compound 11at 0.015 μM when bound to different concentrations of calf thymus (2A),Micrococcus lysodeikticus (2B), Clostridium perfringens (2C) rRNA, DNAand RNA+DNA in solution. FIG. 2 is an overlay of three graphs with theconcentration of RNA 0-150 ng/mL, the concentration of DNA 150-0 ng/mL(along the same axis) and the concentration of RNA+DNA wherein thecombined concentration is always 150 ng/mL where the individualconcentration of RNA and DNA depends on the corresponding concentrationindicated on the axis. In this way the concentrations were combined inthe following format: RNA+DNA respectively, 0 ng/mL+150 ng/mL, 25ng/mL+125 ng/mL, 50 ng/mL+100 ng/mL, 75 ng/mL+75 ng/mL, 100 ng/mL+50ng/mL, 125 ng/mL+25 ng/mL and 150 ng/mL+0 ng/mL. These results indicatethat in solution Compound 11 either does not bind DNA or binds DNA withlittle to no fluorescent signal intensity which is confirmed with a nearzero fluorescence with DNA alone and the same fluorescence intensitysignal for the RNA+DNA as for the corresponding RNA concentration. See,Example 36

FIG. 3: Shows the difference in signal intensity from S. aureus cellstreated with and without rifampicin when loaded with Compound 6 at threedifferent concentrations (0.25, 0.5 and 1 μM). These results demonstratethat Compound 6 binds to both RNA and DNA, but demonstrates a strongersignal intensity when bound to RNA. Compound 6 could be preferentiallybinding to RNA or the compound may be fluorescing brighter on RNA butstill have equal selectively for RNA and DNA. See, Example 37

FIG. 4: Shows the binding of Compound 6 to DNA and RNA in methanol fixedcells in the cytoplasm, nucleus and nucleolus, that were untreated,RNase or DNase treated. RNase treated cells showed a significantreduction in signal intensity while DNase treated cells showed nodecrease in signal intensity indicating that much of the signal seen inuntreated cells is from Compound 6 fluorescing when bound to RNA. See,Example 38

FIG. 5: Shows the binding of Compound 6 (5A) (10 μM) and Compound 19(5B) (1 μM) in formaldehyde fixed cells in the cytoplasm, nucleus andnucleolus, that were untreated, RNase, DNase or RNase and DNase treated.These results show that RNase washing eliminated Compound 6 and Compound19 labeling of the nucleolus (where RNA is most strongly localized) andthat DNase significantly reduced the signal intensity from bothcompounds in this organelle. Both RNase and DNase significantly reducednuclear labeling with Compound 6 whereas nuclear labeling with Compound19 was significantly reduced with RNase, but not DNase. For Compound 6,both RNase and DNase significantly reduced cytoplasmic label, while forCompound 19, cytoplasmic label was not reduced at all with either RNaseor DNase washing. See, Example 39

FIG. 6: Shows detection of intracellular nucleic acid with Compound 11and Compound 6 with peak emission at a concentration of 500 nM for bothcompounds. See, Example 42

DETAILED DESCRIPTION OF THE INVENTION Definitions

Before describing the present invention in detail, it is to beunderstood that this invention is not limited to specific compositionsor process steps, as such may vary. It must be noted that, as used inthis specification and the appended claims, the singular form “a”, “an”and “the” include plural referents unless the context clearly dictatesotherwise. Thus, for example, reference to “a nucleic acid” includes aplurality of nucleic acids and reference to “a reporter molecule”includes a plurality of compounds and the like.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention is related. The following terms aredefined for purposes of the invention as described herein.

Certain compounds of the present invention can exist in unsolvated formsas well as solvated forms, including hydrated forms. In general, thesolvated forms are equivalent to unsolvated forms and are encompassedwithin the scope of the present invention. Certain compounds of thepresent invention may exist in multiple crystalline or amorphous forms.In general, all physical forms are equivalent for the uses contemplatedby the present invention and are intended to be within the scope of thepresent invention.

Certain compounds of the present invention possess asymmetric carbonatoms (optical centers) or double bonds; the racemates, diastereomers,geometric isomers and individual isomers are encompassed within thescope of the present invention.

The compounds of the invention may be prepared as a single isomer (e.g.,enantiomer, cis-trans, positional, diastereomer) or as a mixture ofisomers. In a preferred embodiment, the compounds are prepared assubstantially a single isomer. Methods of preparing substantiallyisomerically pure compounds are known in the art. For example,enantiomerically enriched mixtures and pure enantiomeric compounds canbe prepared by using synthetic intermediates that are enantiomericallypure in combination with reactions that either leave the stereochemistryat a chiral center unchanged or result in its complete inversion.Alternatively, the final product or intermediates along the syntheticroute can be resolved into a single stereoisomer. Techniques forinverting or leaving unchanged a particular stereocenter, and those forresolving mixtures of stereoisomers are well known in the art and it iswell within the ability of one of skill in the art to choose anappropriate method for a particular situation. See, generally, Furnisset al. (eds.) VOGEL'S ENCYCLOPEDIA OF PRACTICAL ORGANIC CHEMISTRY 5^(TH)ED., Longman Scientific and Technical Ltd., Essex, 1991, pp. 809-816;and Heller, Acc. Chem. Res. 23: 128 (1990).

The compounds of the present invention may also contain unnaturalproportions of atomic isotopes at one or more of the atoms thatconstitute such compounds. For example, the compounds may beradiolabeled with radioactive isotopes, such as for example tritium(³H), iodine-125 (¹²⁵I) or carbon-14 (¹⁴C). All isotopic variations ofthe compounds of the present invention, whether radioactive or not, areintended to be encompassed within the scope of the present invention.

Where substituent groups are specified by their conventional chemicalformulae, written from left to right, they equally encompass thechemically identical substituents, which would result from writing thestructure from right to left, e.g., —CH₂O— is intended to also recite—OCH₂—.

The term “affinity” as used herein refers to the strength of the bindinginteraction of two molecules, such as a nucleic acid polymer and anintercalating agent or a positively charged moiety and a negativelycharged moiety.

The terms “alkoxy,” “alkylamino” and “alkylthio” (or thioalkoxy) areused in their conventional sense, and refer to those alkyl groupsattached to the remainder of the molecule via an oxygen atom, an aminogroup, or a sulfur atom, respectively.

The term “alkyl” as used herein refers to a straight, branched or cyclichydrocarbon chain fragment containing between about one and about twentyfive carbon atoms (e.g. methyl, ethyl and the like). Straight, branchedor cyclic hydrocarbon chains having eight or fewer carbon atoms willalso be referred to herein as “lower alkyl”. In addition, the term“alkyl” as used herein further includes one or more substitutions at oneor more carbon atoms of the hydrocarbon chain fragment. Suchsubstitutions include, but are not limited to: aryl; heteroaryl;halogen; alkoxy; amine (—NR′R″); carboxy and thio.

The term “amino” or “amine group” refers to the group —NR′R″ (or NRR′R″)where R, R′ and R″ are independently selected from the group consistingof hydrogen, alkyl, substituted alkyl, aryl, substituted aryl, arylalkyl, substituted aryl alkyl, heteroaryl, and substituted heteroaryl. Asubstituted amine being an amine group wherein R′ or R″ is other thanhydrogen. In a primary amino group, both R′ and R″ are hydrogen, whereasin a secondary amino group, either, but not both, R′ or R″ is hydrogen.In addition, the terms “amine” and “amino” can include protonated andquaternized versions of nitrogen, comprising the group —NRR′R″ and itsbiologically compatible anionic counterions.

The term “aryl” as used herein refers to cyclic aromatic carbon chainhaving twenty or fewer carbon atoms, e.g., phenyl, naphthyl, biphenyl,and anthracenyl. One or more carbon atoms of the aryl group may also besubstituted with, e.g., alkyl; aryl; heteroaryl; a halogen; nitro;cyano; hydroxyl, alkoxyl or aryloxyl; thio or mercapto, alkyl-, orarylthio; amino, alkylamino, arylamino, dialkyl-, diaryl-, orarylalkylamino; aminocarbonyl, alkylaminocarbonyl, arylaminocarbonyl,dialkylaminocarbonyl, diarylaminocarbonyl, or arylalkylaminocarbonyl;carboxyl, or alkyl- or aryloxycarbonyl; aldehyde; aryl- oralkylcarbonyl; iminyl, or aryl- or alkyliminyl; sulfo; alkyl- oralkylcarbonyl; iminyl, or aryl- or alkyliminyl; sulfo; alkyl- orarylsulfonyl; hydroximinyl, or aryl- or alkoximinyl. In addition, two ormore alkyl or heteroalkyl substituents of an aryl group may be combinedto form fused aryl-alkyl or aryl-heteroalkyl ring systems (e.g.,tetrahydronaphthyl). Substituents including heterocyclic groups (e.g.,heteroaryloxy, and heteroaralkylthio) are defined by analogy to theabove-described terms.

The term “aqueous solution” as used herein refers to a solution that ispredominantly water and retains the solution characteristics of water.Where the aqueous solution contains solvents in addition to water, wateris typically the predominant solvent.

The term “complex” as used herein refers to the association of two ormore molecules, usually by non-covalent bonding.

The term “cyanine monomer” or “cyanine dye” as used herein refers to afluorogenic compound that comprises 1) a substituted benzazolium moiety,2) a polymethine bridge and 3) a substituted or unsubstituted pyridiniumor quinolinium moiety. These monomer or dye moieties are capable offorming a non-covalent complex with nucleic acid and demonstrating anincreased fluorescent signal after formation of the nucleic acid-dyecomplex.

The term “detectable response” as used herein refers to a change in oran occurrence of, a signal that is directly or indirectly detectableeither by observation or by instrumentation. Typically, the detectableresponse is an optical response resulting in a change in the wavelengthdistribution patterns or intensity of absorbance or fluorescence or achange in light scatter, fluorescence lifetime, fluorescencepolarization, or a combination of the above parameters.

The term “heteroaryl” as used herein refers to an aryl group as definedabove in which one or more carbon atoms have been replaced by anon-carbon atom, especially nitrogen, oxygen, or sulfur. For example,but not as a limitation, such groups include furyl, tetrahydrofuryl,pyrrolyl, pyrrolidinyl, thienyl, tetrahydrothienyl, oxazolyl,isoxazolyl, triazolyl, thiazolyl, isothiazolyl, pyrazolyl,pyrazolidinyl, oxadiazolyl, thiadiazolyl, imidazolyl, imidazolinyl,pyridyl, pyridaziyl, triazinyl, piperidinyl, morpholinyl,thiomorpholinyl, pyrazinyl, piperainyl, pyrimidinyl, naphthyridinyl,benzofuranyl, benzothienyl, indolyl, indolinyl, indolizinyl, indazolyl,quinolizinyl, qunolinyl, isoquinolinyl, cinnolinyl, phthalazinyl,quinazolinyl, quinoxalinyl, pteridinyl, quinuclidinyl, carbazolyl,acridinyl, phenazinyl, phenothizinyl, phenoxazinyl, purinyl,benzimidazolyl and benzthiazolyl and their aromatic ring-fused analogs.Many fluorophores are comprised of heteroaryl groups and include,without limitations, xanthenes, oxazines, benzazolium derivatives(including cyanines and carbocyanines), borapolyazaindacenes,benzofurans, indoles and quinazolones.

The above heterocyclic groups may further include one or moresubstituents at one or more carbon and/or non-carbon atoms of theheteroaryl group, e.g., alkyl; aryl; heterocycle; halogen; nitro; cyano;hydroxyl, alkoxyl or aryloxyl; thio or mercapto, alkyl- or arylthio;amino, alkyl-, aryl-, dialkyl-, diaryl-, or arylalkylamino;aminocarbonyl, alkylaminocarbonyl, arylaminocarbonyl,dialkylaminocarbonyl, diarylaminocarbonyl or arylalkylaminocarbonyl;carboxyl, or alkyl- or aryloxycarbonyl; aldehyde; aryl- oralkylcarbonyl; iminyl, or aryl- or alkyliminyl; sulfo; alkyl- orarylsulfonyl; hydroximinyl, or aryl- or alkoximinyl. In addition, two ormore alkyl substituents may be combined to form fused heterocycle-alkylring systems. Substituents including heterocyclic groups (e.g.,heteroaryloxy, and heteroaralkylthio) are defined by analogy to theabove-described terms.

The term “heterocycloalkyl” as used herein refers to a heterocycle groupthat is joined to a parent structure by one or more alkyl groups asdescribed above, e.g., 2-piperidylmethyl, and the like. The term“heterocycloalkyl” refers to a heteroaryl group that is joined to aparent structure by one or more alkyl groups as described above, e.g.,2-thienylmethyl, and the like.

The term “kit” as used refers to a packaged set of related components,typically one or more compounds or compositions.

The term “nucleic acid polymer” as used herein refers to natural orsynthetic polymers of DNA or RNA that are single, double, triple orquadruple stranded. Polymers are two or more bases in length.

The term “nucleic acid reporter molecule” as used herein refers to thepresent dimer compounds represented by the formula A-L-B wherein L is alinker comprising at least one aromatic, heteroaromatic, cyclic orheterocyclic moiety and A and B are nucleic acid intercalating monomercompounds, which may be the same or different.

The term “reactive group” as used herein refers to a group that iscapable of reacting with another chemical group to form a covalent bond,i.e. is covalently reactive under suitable reaction conditions, andgenerally represents a point of attachment for another substance. Thereactive group is a moiety, such as carboxylic acid or succinimidylester, on the compounds of the present invention that is capable ofchemically reacting with a functional group on a different compound toform a covalent linkage. Reactive groups generally include nucleophiles,electrophiles and photoactivatable groups.

Exemplary reactive groups include, but are not limited to, olefins,acetylenes, alcohols, phenols, ethers, oxides, halides, aldehydes,ketones, carboxylic acids, esters, amides, cyanates, isocyanates,thiocyanates, isothiocyanates, amines, hydrazines, hydrazones,hydrazides, diazo, diazonium, nitro, nitriles, mercaptans, sulfides,disulfides, sulfoxides, sulfones, sulfonic acids, sulfinic acids,acetals, ketals, anhydrides, sulfates, sulfenic acids isonitriles,amidines, imides, imidates, nitrones, hydroxylamines, oximes, hydroxamicacids thiohydroxamic acids, allenes, ortho esters, sulfites, enamines,ynamines, ureas, pseudoureas, semicarbazides, carbodiimides, carbamates,imines, azides, azo compounds, azoxy compounds, and nitroso compounds.Reactive functional groups also include those used to preparebioconjugates, e.g., N-hydroxysuccinimide esters, maleimides and thelike. Methods to prepare each of these functional groups are well knownin the art and their application to or modification for a particularpurpose is within the ability of one of skill in the art (see, forexample, Sandler and Karo, eds. ORGANIC FUNCTIONAL GROUP PREPARATIONS,Academic Press, San Diego, 1989).

The term “reporter molecule” as used herein refers to any luminescentmolecule that is capable of associating with a nucleic acid polymer andproducing a detectable signal. Typically, reporter molecules includeunsymmetrical cyanine dyes, dimmers of cyanine dyes, ethidium bromide,DAPI, Hoechst, acridine and styryl dyes that are capable of producing adetectable signal upon appropriate wavelength excitation.

The term “sample” as used herein refers to any material that may containa target ligand. Typically, the sample is a live cell, a biologicalfluid that comprises endogenous host cell proteins, nucleic acidpolymers, nucleotides, oligonucleotides, peptides and buffer solutions.The sample may be in an aqueous solution, a viable cell culture orimmobilized on a solid or semi solid surface such as a polyacrylamidegel, membrane blot or on a microarray.

Compounds

In general, for ease of understanding the present invention, the nucleicacid reporter molecules and corresponding substituents will first bedescribed in detail, followed by the many and varied methods in whichthe compounds find uses, which is followed by exemplified methods of useand synthesis of novel compounds that are particularly advantageous foruse with the methods of the present invention.

In one embodiment the present invention provides novel linkers thatcomprise at least one aromatic, heteroaromatic, cyclic or heterocyclicmoiety that covalently bonds two nucleic acid complexing monomercompounds into a hetero- or homodimer that form nucleic acid reportermolecules. Without wishing to be bound by a theory, it appears thattypically the complexing monomer compounds bind in the minor groove ofnucleic acid, but compounds that bind in the major groove are alsoincluded. The aromatic, heteroaromatic, cyclic or heterocyclic moietytypically comprises 3-20 non-hydrogen atoms selected from the groupconsisting of N, O, P, C and S. The nucleic acid complexing compoundsinclude, without limitation, any compound known to one skilled in theart and novel compounds yet to be discovered, such as cyanine dyes,styryl dyes, ethidium bromide, DAPI, Hoechst and acridine. There is nointended limitation on the nucleic acid complexing compound.

In this instance present nucleic acid reporter molecules are representedby the general formula A-L-B wherein A and B, which may be the same ordifferent, are nucleic acid complexing compound monomers and L is alinker that comprises at least one aromatic, heteroaromatic, cyclic orheterocyclic moiety. These compounds may be cell permeable or non-cellpermeable depending on the lipophilic properties of the individualcompounds.

Typically, the nucleic acid complexing compound monomers areunsymmetrical cyanine dyes including, but are not limited to, dyes soldunder the trade name SYBR® dyes (Molecular Probes, Inc.), thiazoleorange, their derivatives and any monomer compound disclosed in U.S.Pat. Nos. 4,957,870; 4,883,867; 5,436,134; 5,658,751, 5,534,416 and5,863,753.

Thus, in one aspect the compounds of the present invention include, butare not limited to, a nucleic acid reporter molecule comprising twounsymmetrical cyanine monomer moieties which may be the same ordifferent, covalently attached by a linker comprising at least onearomatic, heteroaromatic, cyclic or heterocyclic moiety comprising 3-20non-hydrogen atoms selected from the group consisting of O, N, S, P andC.

In another exemplary embodiment, the present dimer compounds are cellpermeable due to the lipophilicity of the compounds. These compounds arerepresented by the general formula A-L*-B wherein A and B, which may bethe same or different, are nucleic acid compound monomers that are cellpermeable unsymmetrical cyanine dyes, and the linker L* is a series ofstable covalent bonds the linker typically incorporates 1-30 nonhydrogenatoms selected from the group consisting of C, N, O, S and P. This classof compounds represent, for the first time, cyanine dimers that are cellpermeable. While the monomers are lipophilic it is not obvious, due tothe size of the compound, that these dimer compounds would be cellpermeable. Thus, these compounds represent an improvement over knowndimer compounds, which are not cell permeable and thus unable toassociate with and detect nucleic acid in a live cell.

1. Unsymmetrical Cyanine Monomer Moieties

Typically, A and B are unsymmetrical cyanine monomer moieties. Thecyanine monomer moieties are further comprised of three moieties; F-M-K,wherein the F moiety is a substituted or unsubstituted benzazolium ringsystem that may or may not contain a quarternized nitrogen atom, the Mmoiety is a mono or polymethine bridge and the K moiety is substitutedor unsubstituted pyridinium or quinolinium moiety.

The unsymmetrical cyanine moieties are typically represented by thegeneral formula:

wherein R³ is selected from the group consisting of hydrogen, C₁-C₆alkyl, C₁-C₆ alkoxy, fused benzene, trifluoromethyl, halogen, reactivegroup, carrier molecule and solid support, each of which may beoptionally further substituted. R³ is typically hydrogen, butsubstituents other than hydrogen may used to alter the absorption andemission spectra of the compound. In addition, the benzazolium moietymay contain more that one R³, (1-4), which may be the same or different.But, typically there is not more than one R³ that is not hydrogen.

R⁴ is a C₁-C₆ alkyl, a reactive group, a carrier molecule or a solidsupport and X is O, S, or CR⁶R⁷ wherein R⁶ and R⁷ are independently aC₁-C₆ alkyl group, a reactive group, carrier molecule, solid support ortaken together form a 5- or 6-membered saturated ring, each of which maybe further substituted. Typically, R⁴ is methyl or ethyl preferably R⁴is methyl.

The methine bridge consists of 1, 3 or 5 methine groups (—CH═), whereinJ is 0, 1 or 2; that covalently attaches the benzazolium moiety to the Kmoiety of the cyanine monomer compound. The length of the methine bridgehas a considerable effect on the absorption and emission spectra of theunsymmetrical cyanine monomer moieties. In addition, we have found thatmonomethine cyanine monomer moieties when comprising a present nucleicacid reporter molecule that is attached by a rigid linker according toFormula (II) fluoresce more intensely on RNA than DNA when in thepresence of DNA. In this instance, when complexed with nucleic acid (RNAor DNA) the ratio of fluorescence enhancement (fluorescent intensitysignal) is greater than one for RNA compared to DNA. Thus, monomethineunsymmetrical cyanine monomer moieties with rigid linkers are preferredfor RNA reporter molecules.

In one aspect of the invention, R³ is hydrogen, R⁴ is methyl, X is S andt is 0. In another aspect of the invention R³ is hydrogen, R⁴ is methyl,X is O and t is 0. In yet another aspect, t is 1.

The K moiety is a substituted or unsubstituted pyridinium or quinoliniummoiety. The substitutents of the pyridinium or quinolinium moietyinclude substituents well known in the art, including hydrogen, alkylgroup, halogen, alkoxy, a saturated or unsaturated, substituted orunsubstituted cyclic substituent including aryl, benzene, phenyl group,reactive group, carrier molecule or solid support. Typically, thepyridinium or quinolinium moiety is substituted by hydrogen or alkoxy ata ring carbon atom. Preferably the alkoxy group is methoxy.

A C₁-C₆ alkyl group or a saturated or unsaturated, substituted orunsubstituted cyclic substituent typically substitutes the nitrogen atomof the pyridinium or quinolinium moiety. The alkyl group and cyclicsubstituent may be further substituted by hydrogen, alkyl, amino,alkylamino, alkoxy or carboxyalkyl. Typically, a methyl group or anaromatic group such as phenyl substitutes the nitrogen atom. Withoutwishing to be bound by a theory it appears that the cyclic substituentincreases the membrane permeability of the cyanine monomer moieties andis, thus, typically included for present nucleic acid reporter moleculesthat are to be used to detect nucleic acid in live or fixed cells.

The pyridinium or quinolinium moiety is also substituted by the presentlinker wherein the linker attaches two cyanine monomer moieties at theirrespective K moieties. The dimer compounds may be homo- or heterodimercompounds wherein the unsymmetrical cyanine monomer moieties may be thesame or different.

In one aspect of the invention, a preferred unsymmetrical cyaninemonomer is represented by the general formula:

The dashed line represents the point of attachment for the linker on thequinolinium moiety and X is S or O. For simplicity the benzosubstitutents are represented by hydrogen but the benzo rings may besubstituted by any moiety known to one skilled in the art, including,but not limited to C₁-C₆ alkyl, C₁-C₆ alkoxy, fused benzene,trifluoromethyl, halogen, reactive group, carrier molecule or solidsupport. These substituents may be further substituted, as describedabove.

In another aspect of the invention, a cyanine monomer is represented bythe general formula:

wherein the pyridinium moiety is substituted by a phenyl group on thenitrogen atom. The dashed line represents the point of attachment forthe linker and X is S or O.

Alternatively, the following compound is also preferred wherein t isrepresented by 1, resulting in a trimethine cyanine monomer moietyrepresented by the general formula:

In another aspect of the invention, the unsymmetrical cyanine monomer isrepresented by the general formula:

wherein the nitrogen atom of the quinolinium moiety is substituted by analkyl group and the dashed line represents the point of attachment forthe linker and X is O or S.

In yet another aspect of the invention, the unsymmetrical cyaninemonomer is represented by the general formula:

Wherein the quinolinium moiety is substituted by a C₁-C₆ alkoxy whereink is 0-5. Typically, k is 0 designating a methoxy group. The dashed linerepresents the point of attachment for the linker and X is O or S.

In an additional example of the invention, a julolidine derivative isemployed and is represented by the general formula:

Wherein the dashed line represents the point of attachment for thelinker and X is O or S.

In another example of the invention, the methylated quinolium isattached ortho to the cyanine monomer.

Wherein the dashed line represents the point of attachment for thelinker and X is C(CH₃)₂.

In an exemplified embodiment of the present invention, a phenylsubstituted quinolium attached para to an indolenine monomer isrepresent by the general formula:

Wherein the dashed line represents the point of attachment for thelinker.

2. Linkers

The present nucleic acid reporter molecules comprise a linker covalentlyattaching two monomer moieties wherein the linker (L) comprises at leastone aromatic, heteroaromatic, cyclic or heterocyclic moiety. The ringmoiety contains 3-20 non-hydrogen atoms selected from the groupconsisting of C, N, O, S, and P. This moiety in its self may conferrigidity to the linker, or when part of longer linker is typically notrigid. These linkers have novel properties in forming unsymmetricalcyanine dimer compounds and provide nucleic acid reporter molecules withunexpected advantages for detecting RNA in the presence of DNA.

Typically the linker is represented by Formula (I)—(Y)_(r)—(CH₂)_(m)-T_(q)-(CH₂)_(n)-E-(CH₂)_(n)-T_(q)-(CH₂)_(m)—(Y)_(r)—.When present, Y is a linear, branched, cyclic or aromatic moietycomprising 1-20 non-hydrogen atoms selected from the group consisting ofC, N, O, P and S. The Y moiety may be any combination of stable chemicalbonds, optionally including, single, double, triple or aromaticcarbon-carbon bonds, as well as carbon-nitrogen bonds, nitrogen-nitrogenbonds, carbon-oxygen bonds, sulfur-sulfur bonds, carbon-sulfur bonds,phosphorus-oxygen bonds, phosphorus-nitrogen bonds, andnitrogen-platinum bonds. Typically the Y moiety incorporates less than15 nonhydrogen atoms and are composed of any combination of ether,thioether, thiourea, amine, ester, carboxamide, sulfonamide, hydrazidebonds and aromatic or heteroaromatic bonds. Typically the Y moiety is acombination of single carbon-carbon bonds and carboxamide, sulfonamideor thioether bonds. The bonds of the Y moiety typically result in thefollowing moieties that can be found in the Y moiety: ether, thioether,carboxamide, thiourea, sulfonamide, urea, urethane, hydrazine, alkyl,aryl, heteroaryl, alkoxy, cycloalkyl and amine moieties. The Y moietymay be absent or present wherein r is 0 or 1. In addition, the Y moietymay be substituted by a reactive group, carrier molecule or solidsupport.

T is a heteroatom selected from the group consisting of P, O, S, NR²wherein R² is hydrogen, amine, substituted amine, a C₁-C₆ alkyl group,reactive group, carrier molecule, solid support or is absent. Typically,the heteroatom is S, NR² or is absent wherein q is 0, when present qis 1. Preferably the heteroatom is NR². The alkyl group —(CH₂)— directlyadjacent to the E moiety may be independently a methyl group or absentwherein n is 0 or 1. The alkyl group between the Y moiety and the Theteroatom may independently be absent or a C₁-C₆ alkyl.

The E moiety is an aromatic, heteroaromatic, cyclic or heterocyclicmoiety comprising 3-20 non-hydrogen atoms selected from the groupconsisting of O, N, S, P and C. The E moiety can be fully saturated orunsaturated and can contain no heteroatoms or one or more heteroatoms.Typically, the heteroatoms, when present, are oxygen or nitrogen. The Emoiety typically includes, without limitation, benzene, pyrimidine,piperazine, piperidine, cyclohexane, cyclopentane, dioxane,tetrahydropyran, tetrahydrofuran, pyrole, thiophene, furna, oxazole,pyridine, thiazole, cyclen and pyrrolidine.

Thus, in one aspect of the invention, the E moiety is selected from thegroup consisting of

Wherein R² is hydrogen, amine, substituted amine, substituted orunsubstituted C₁-C₆ alkyl, unsubstituted reactive group, substitutedreactive group, unsubstituted carrier molecule, substituted carriermolecule, unsubstituted solid support or substituted solid support.

In one aspect of the invention, the linkers are rigid and arerepresented by Formula (II)—(CH₂)_(n)-T_(q)-(CH₂)_(n)-E-(CH₂)_(n)-T_(q)-(CH₂)_(n)— wherein T and Eare as described above. n and q are independently 0 or 1. These linkersare not permitted to have a long alkyl chain or the Y moiety.

Preferred rigid linkers are according to the following general formulaswherein the E moiety is designated:

Each R⁸, R⁹, R¹⁰, R¹¹ and R¹² are independently a substituted orunsubstituted heteroatom and each Z is independently methylene or isabsent that is represented as —(CH₂)_(g) wherein g is 0 or 1. The dashedline is attached directly to the respective K moiety of theunsymmetrical cyanine monomer. Typically, the heteroatom is —NR², —N═ orS wherein R² is hydrogen, amine, substituted amine, substituted orunsubstituted C₁-C₆ alkyl, amine, substituted amine, unsubstitutedreactive group, substituted reactive group, unsubstituted carriermolecule, substituted carrier molecule, unsubstituted solid support orsubstituted solid support.

In a further embodiment, formula (II)(i) is represented by the linkers

Thus, selected linkers include:

As discussed above, in one aspect of the invention, the present nucleicacid reporter molecules are used to detect RNA in the presence of DNA.In this aspect the linkers are typically selected from the groupconsisting of

When these linkers attach two monomethine unsymmetrical cyanine monomermoieties in a present nucleic acid reporter molecule the molecule formsa nucleic acid-reporter molecule complex that is capable of fluorescingmore intensely on RNA than DNA.

Alternatively, the present compounds comprise the linker (L*), whereinthe linker is a series of stable covalent bonds. The linker typicallyincorporates 1-30 nonhydrogen atoms selected from the group consistingof C, N, O, S and P. The linker may be any combination of stablechemical bonds, optionally including, single, double, triple or aromaticcarbon-carbon bonds, as well as carbon-nitrogen bonds, nitrogen-nitrogenbonds, carbon-oxygen bonds, sulfur-sulfur bonds, carbon-sulfur bonds,phosphorus-oxygen bonds, phosphorus-nitrogen bonds, andnitrogen-platinum bonds. Typically the linker incorporates less than 15nonhydrogen atoms and are composed of any combination of ether,thioether, thiourea, amine, ester, carboxamide, sulfonamide, hydrazidebonds and aromatic or heteroaromatic bonds. Typically the linker is acombination of single carbon-carbon bonds and carboxamide, sulfonamideor thioether bonds. The bonds of the linker typically result in thefollowing moieties that can be found in the linker: ether, thioether,carboxamide, thiourea, sulfonamide, urea, urethane, hydrazine, alkyl,aryl, heteroaryl, alkoxy, cycloalkyl and amine moieties. Examples of alinker include substituted or unsubstituted polymethylene, arylene,alkylarylene, arylenealkyl, and arylthio.

In one embodiment, the linker contains 1-6 carbon atoms; in another, thelinker comprises a thioether linkage. Exemplary linking members includea moiety that includes —C(O)NH—, —C(O)O—, —NH—, —S—, —O—, and the like.In another embodiment, the linker is or incorporates the formula—(CH₂)_(d)(CONH(CH₂)_(e))_(z)— or where d is an integer from 0-5, e isan integer from 1-5 and z is 0 or 1. In a further embodiment, the linkeris or incorporates the formula —O—(CH₂)—. In yet another embodiment, thelinker is or incorporates a phenylene or a 2-carboxy-substitutedphenylene.

Reactive Groups, Carrier Molecules and Solid Supports

The present compounds, in certain embodiments, are chemically reactivewherein the compounds comprise a reactive group. In a furtherembodiment, the compounds comprise a carrier molecule or solid support.These substituents, reactive groups, carrier molecules, and solidsupports, comprise a linker that is used to covalently attach thesubstituents to any of the moieties of the present compounds having theformula A-L-B or A-L*-B. The solid support, carrier molecule or reactivegroup may be directly attached (where linker is a single bond) to themoieties or attached through a series of stable bonds, as disclosedabove.

Any combination of linkers may be used to attach the carrier molecule,solid support or reactive group and the present compounds together. Thelinker may also be substituted to alter the physical properties of thereporter moiety or chelating moiety, such as spectral properties of thedye.

An important feature of the linker is to provide an adequate spacebetween the carrier molecule, reactive group or solid support and thenucleic acid reporter molecule so as to prevent steric hinderance.Therefore, the linker of the present compound is important for (1)attaching the carrier molecule, reactive group or solid support to thecompound, (2) providing an adequate space between the carrier molecule,reactive group or solid support and the compound so as not to stericallyhinder the action of the compound and (3) for altering the physicalproperties of the present compounds.

In another exemplary embodiment of the invention, the present compoundsare chemically reactive, and are substituted by at least one reactivegroup. The reactive group functions as the site of attachment foranother moiety, such as a carrier molecule or a solid support, whereinthe reactive group chemically reacts with an appropriate reactive orfunctional group on the carrier molecule or solid support. Thus, inanother aspect of the present invention the compounds comprise thechelating moiety, linker, reporter moiety, a reactive group moiety andoptionally a carrier molecule and/or a solid support.

In an exemplary embodiment, the compounds of the invention furthercomprise a reactive group which is a member selected from an acrylamide,an activated ester of a carboxylic acid, a carboxylic ester, an acylazide, an acyl nitrile, an aldehyde, an alkyl halide, an anhydride, ananiline, an amine, an aryl halide, an azide, an aziridine, a boronate, adiazoalkane, a haloacetamide, a haloalkyl, a halotriazine, a hydrazine,an imido ester, an isocyanate, an isothiocyanate, a maleimide, aphosphoramidite, a photoactivatable group, a reactive platinum complex,a silyl halide, a sulfonyl halide, and a thiol. In a particularembodiment the reactive group is selected from the group consisting ofcarboxylic acid, succinimidyl ester of a carboxylic acid, hydrazide,amine and a maleimide. In exemplary embodiment, at least one memberselected from A, L or B comprises a reactive group. Preferably, at leastone of A or B comprises a reactive group, wherein at least one of R³,R⁴, R⁶ or R⁷ is a reactive group or is attached to a reactive group. Inanother aspect, L comprises a reactive group; typically the E moiety isattached to a reactive group. Alternatively, if the present compoundcomprises a carrier molecule or solid support a reactive group may becovalently attached independently to those substituents, allowing forfurther conjugation to a reporter molecule, carrier molecule or solidsupport.

In one aspect, the compound comprises at least one reactive group thatselectively reacts with an amine group. This amine-reactive group isselected from the group consisting of succinimidyl ester, sulfonylhalide, tetrafluorophenyl ester and iosothiocyanates. Thus, in oneaspect, the present compounds form a covalent bond with anamine-containing molecule in a sample. In another aspect, the compoundcomprises at least one reactive group that selectively reacts with athiol group. This thiol-reactive group is selected from the groupconsisting of maleimide, haloalkyl and haloacetamide (including anyreactive groups disclosed in U.S. Pat. Nos. 5,362,628; 5,352,803 and5,573,904).

The pro-reactive groups are synthesized during the formation of themonomer moieties and carrier molecule and solid support containingcompounds to provide chemically reactive nucleic acid reportercompounds. In this way, compounds incorporating a reactive group can becovalently attached to a wide variety of carrier molecules or solidsupports that contain or are modified to contain functional groups withsuitable reactivity, resulting in chemical attachment of the components.In an exemplary embodiment, the reactive group of the compounds of theinvention and the functional group of the carrier molecule or solidsupport comprise electrophiles and nucleophiles that can generate acovalent linkage between them. Alternatively, the reactive groupcomprises a photoactivatable group, which becomes chemically reactiveonly after illumination with light of an appropriate wavelength.Typically, the conjugation reaction between the reactive group and thecarrier molecule or solid support results in one or more atoms of thereactive group being incorporated into a new linkage attaching thepresent compound of the invention to the carrier molecule or solidsupport. Selected examples of functional groups and linkages are shownin Table 1, where the reaction of an electrophilic group and anucleophilic group yields a covalent linkage.

TABLE 1 Examples of some routes to useful covalent linkagesElectrophilic Group Nucleophilic Group Resulting Covalent Linkageactivated esters* amines/anilines carboxamides acrylamides thiolsthioethers acyl azides** amines/anilines carboxamides acyl halidesamines/anilines carboxamides acyl halides alcohols/phenols esters acylnitriles alcohols/phenols esters acyl nitriles amines/anilinescarboxamides aldehydes amines/anilines imines aldehydes or ketoneshydrazines hydrazones aldehydes or ketones hydroxylamines oximes alkylhalides amines/anilines alkyl amines alkyl halides carboxylic acidsesters alkyl halides thiols thioethers alkyl halides alcohols/phenolsethers alkyl sulfonates thiols thioethers alkyl sulfonates carboxylicacids esters alkyl sulfonates alcohols/phenols ethers anhydridesalcohols/phenols esters anhydrides amines/anilines carboxamides arylhalides thiols thiophenols aryl halides amines aryl amines aziridinesthiols thioethers boronates glycols boronate esters carbodiimidescarboxylic acids N-acylureas or anhydrides diazoalkanes carboxylic acidsesters epoxides thiols thioethers haloacetamides thiols thioethershaloplatinate amino platinum complex haloplatinate heterocycle platinumcomplex haloplatinate thiol platinum complex halotriazinesamines/anilines aminotriazines halotriazines alcohols/phenols triazinylethers halotriazines thiols triazinyl thioethers imido estersamines/anilines amidines isocyanates amines/anilines ureas isocyanatesalcohols/phenols urethanes isothiocyanates amines/anilines thioureasmaleimides thiols thioethers phosphoramidites alcohols phosphite esterssilyl halides alcohols silyl ethers sulfonate esters amines/anilinesalkyl amines sulfonate esters thiols thioethers sulfonate esterscarboxylic acids esters sulfonate esters alcohols ethers sulfonylhalides amines/anilines sulfonamides sulfonyl halides phenols/alcoholssulfonate esters *Activated esters, as understood in the art, generallyhave the formula —COΩ, where Ω is a good leaving group (e.g.,succinimidyloxy (—OC₄H₄O₂) sulfosuccinimidyloxy (—OC₄H₃O₂—SO₃H),-1-oxybenzotriazolyl (—OC₆H₄N₃); or an aryloxy group or aryloxysubstituted one or more times by electron withdrawing substituents suchas nitro, fluoro, chloro, cyano, or trifluoromethyl, or combinationsthereof, used to form activated aryl esters; or a carboxylic acidactivated by a carbodiimide to form an anhydride or mixed anhydride—OCOR^(a) or —OCNR^(a)NHR^(b), where R^(a) and R^(b), which may be thesame or different, are C₁-C₆ alkyl, C₁-C₆ perfluoroalkyl, or C₁-C₆alkoxy; or cyclohexyl, 3-dimethylaminopropyl, or N-morpholinoethyl).**Acyl azides can also rearrange to isocyanates

Choice of the reactive group used to attach the compound of theinvention to the substance to be conjugated typically depends on thereactive or functional group on the substance to be conjugated and thetype or length of covalent linkage desired. The types of functionalgroups typically present on the organic or inorganic substances(biomolecule or non-biomolecule) include, but are not limited to,amines, amides, thiols, alcohols, phenols, aldehydes, ketones,phosphates, imidazoles, hydrazines, hydroxylamines, disubstitutedamines, halides, epoxides, silyl halides, carboxylate esters, sulfonateesters, purines, pyrimidines, carboxylic acids, olefinic bonds, or acombination of these groups. A single type of reactive site may beavailable on the substance (typical for polysaccharides or silica), or avariety of sites may occur (e.g., amines, thiols, alcohols, phenols), asis typical for proteins.

Typically, the reactive group will react with an amine, a thiol, analcohol, an aldehyde, a ketone, or with silica. Preferably, reactivegroups react with an amine or a thiol functional group, or with silica.In one embodiment, the reactive group is an acrylamide, an activatedester of a carboxylic acid, an acyl azide, an acyl nitrile, an aldehyde,an alkyl halide, a silyl halide, an anhydride, an aniline, an arylhalide, an azide, an aziridine, a boronate, a diazoalkane, ahaloacetamide, a halotriazine, a hydrazine (including hydrazides), animido ester, an isocyanate, an isothiocyanate, a maleimide, aphosphoramidite, a reactive platinum complex, a sulfonyl halide, or athiol group. By “reactive platinum complex” is particularly meantchemically reactive platinum complexes such as described in U.S. Pat.No. 5,714,327.

Where the reactive group is an activated ester of a carboxylic acid,such as a succinimidyl ester of a carboxylic acid, a sulfonyl halide, atetrafluorophenyl ester or an isothiocyanates, the resulting compound isparticularly useful for preparing conjugates of carrier molecules suchas proteins, nucleotides, oligonucleotides, or haptens. Where thereactive group is a maleimide, haloalkyl or haloacetamide (including anyreactive groups disclosed in U.S. Pat. Nos. 5,362,628; 5,352,803 and5,573,904 (supra)) the resulting compound is particularly useful forconjugation to thiol-containing substances. Where the reactive group isa hydrazide, the resulting compound is particularly useful forconjugation to periodate-oxidized carbohydrates and glycoproteins, andin addition is an aldehyde-fixable polar tracer for cell microinjection.Where the reactive group is a silyl halide, the resulting compound isparticularly useful for conjugation to silica surfaces, particularlywhere the silica surface is incorporated into a fiber optic probesubsequently used for remote ion detection or quantitation.

In a particular aspect, the reactive group is a photoactivatable groupsuch that the group is only converted to a reactive species afterillumination with an appropriate wavelength. An appropriate wavelengthis generally a UV wavelength that is less than 400 nm. This methodprovides for specific attachment to only the target molecules, either insolution or immobilized on a solid or semi-solid matrix.Photoactivatable reactive groups include, without limitation,benzophenones, aryl azides and diazirines.

Preferably, the reactive group is a photoactivatable group, succinimidylester of a carboxylic acid, a haloacetamide, haloalkyl, a hydrazine, anisothiocyanate, a maleimide group, an aliphatic amine, a silyl halide, acadaverine or a psoralen. More preferably, the reactive group is asuccinimidyl ester of a carboxylic acid, a maleimide, an iodoacetamide,or a silyl halide. In a particular embodiment the reactive group is asuccinimidyl ester of a carboxylic acid, a sulfonyl halide, atetrafluorophenyl ester, an iosothiocyanates or a maleimide.

In another exemplary embodiment, the present compound is covalentlybound to a carrier molecule. If the compound has a reactive group, thenthe carrier molecule can alternatively be linked to the compound throughthe reactive group. The reactive group may contain both a reactivefunctional moiety and a linker, or only the reactive functional moiety.

A variety of carrier molecules are useful in the present invention.Exemplary carrier molecules include antigens, steroids, vitamins, drugs,haptens, metabolites, toxins, environmental pollutants, amino acids,peptides, proteins, nucleic acids, nucleic acid polymers, carbohydrates,lipids, and polymers. In exemplary embodiment, at least one memberselected from A, L or B comprises a carrier molecule. Preferably, atleast one of A or B comprises a carrier molecule, wherein at least oneof R³, R⁴, R⁶ or R⁷ is a carrier molecule or is attached to a carriermolecule. In another aspect, L comprises a carrier molecule; typicallythe E moiety is attached to a carrier molecule.

In an exemplary embodiment, the carrier molecule comprises an aminoacid, a peptide, a protein, a polysaccharide, a nucleoside, anucleotide, an oligonucleotide, a nucleic acid, a hapten, a psoralen, adrug, a hormone, a lipid, a lipid assembly, a synthetic polymer, apolymeric microparticle, a biological cell, a virus and combinationsthereof. In another exemplary embodiment, the carrier molecule isselected from a hapten, a nucleotide, an oligonucleotide, a nucleic acidpolymer, a protein, a peptide or a polysaccharide. In a preferredembodiment the carrier molecule is amino acid, a peptide, a protein, apolysaccharide, a nucleoside, a nucleotide, an oligonucleotide, anucleic acid, a hapten, a psoralen, a drug, a hormone, a lipid, a lipidassembly, a tyramine, a synthetic polymer, a polymeric microparticle, abiological cell, cellular components, an ion chelating moiety, anenzymatic substrate or a virus. In another preferred embodiment, thecarrier molecule is an antibody or fragment thereof, an antigen, anavidin or streptavidin, a biotin, a dextran, an IgG binding protein, afluorescent protein, agarose, and a non-biological microparticle.

In an exemplary embodiment, the enzymatic substrate is selected from anamino acid, peptide, sugar, alcohol, alkanoic acid, 4-guanidinobenzoicacid, nucleic acid, lipid, sulfate, phosphate, —CH₂OCOalkyl andcombinations thereof. Thus, the enzyme substrates can be cleave byenzymes selected from the group consisting of peptidase, phosphatase,glycosidase, dealkylase, esterase, guanidinobenzotase, sulfatase,lipase, peroxidase, histone deacetylase, endoglycoceramidase,exonuclease, reductase and endonuclease.

In another exemplary embodiment, the carrier molecule is an amino acid(including those that are protected or are substituted by phosphates,carbohydrates, or C₁ to C₂₂ carboxylic acids), or a polymer of aminoacids such as a peptide or protein. In a related embodiment, the carriermolecule contains at least five amino acids, more preferably 5 to 36amino acids. Exemplary peptides include, but are not limited to,neuropeptides, cytokines, toxins, protease substrates, and proteinkinase substrates. Other exemplary peptides may function as organellelocalization peptides, that is, peptides that serve to target theconjugated compound for localization within a particular cellularsubstructure by cellular transport mechanisms. Preferred protein carriermolecules include enzymes, antibodies, lectins, glycoproteins, histones,albumins, lipoproteins, avidin, streptavidin, protein A, protein G,phycobiliproteins and other fluorescent proteins, hormones, toxins andgrowth factors. Typically, the protein carrier molecule is an antibody,an antibody fragment, avidin, streptavidin, a toxin, a lectin, or agrowth factor. Exemplary haptens include biotin, digoxigenin andfluorophores.

In another exemplary embodiment, the carrier molecule comprises anucleic acid base, nucleoside, nucleotide or a nucleic acid polymer,optionally containing an additional linker or spacer for attachment of afluorophore or other ligand, such as an alkynyl linkage (U.S. Pat. No.5,047,519), an aminoallyl linkage (U.S. Pat. No. 4,711,955) or otherlinkage. In another exemplary embodiment, the nucleotide carriermolecule is a nucleoside or a deoxynucleoside or a dideoxynucleoside.

Exemplary nucleic acid polymer carrier molecules are single- ormulti-stranded, natural or synthetic DNA or RNA oligonucleotides, orDNA/RNA hybrids, or incorporating an unusual linker such as morpholinederivatized phosphates (AntiVirals, Inc., Corvallis Oreg.), or peptidenucleic acids such as N-(2-aminoethyl)glycine units, where the nucleicacid contains fewer than 50 nucleotides, more typically fewer than 25nucleotides.

In another exemplary embodiment, the carrier molecule comprises acarbohydrate or polyol that is typically a polysaccharide, such asdextran, FICOLL, heparin, glycogen, amylopectin, mannan, inulin, starch,agarose and cellulose, or is a polymer such as a poly(ethylene glycol).In a related embodiment, the polysaccharide carrier molecule includesdextran, agarose or FICOLL.

In another exemplary embodiment, the carrier molecule comprises a lipid(typically having 6-25 carbons), including glycolipids, phospholipids,and sphingolipids. Alternatively, the carrier molecule comprises a lipidvesicle, such as a liposome, or is a lipoprotein (see below). Somelipophilic substituents are useful for facilitating transport of theconjugated dye into cells or cellular organelles.

Alternatively, the carrier molecule is a cell, cellular systems,cellular fragment, or subcellular particles, including virus particles,bacterial particles, virus components, biological cells (such as animalcells, plant cells, bacteria, or yeast), or cellular components.Examples of cellular components that are useful as carrier moleculesinclude lysosomes, endosomes, cytoplasm, nuclei, histones, mitochondria,Golgi apparatus, endoplasmic reticulum and vacuoles.

In another exemplary embodiment, the carrier molecule non-covalentlyassociates with organic or inorganic materials. Exemplary embodiments ofthe carrier molecule that possess a lipophilic substituent can be usedto target lipid assemblies such as biological membranes or liposomes bynon-covalent incorporation of the dye compound within the membrane,e.g., for use as probes for membrane structure or for incorporation inliposomes, lipoproteins, films, plastics, lipophilic microspheres orsimilar materials.

In an exemplary embodiment, the carrier molecule comprises a specificbinding pair member wherein the present compounds are conjugated to aspecific binding pair member and are used to detect nucleic acids.Alternatively, the presence of the labeled specific binding pair memberindicates the location of the complementary member of that specificbinding pair; each specific binding pair member having an area on thesurface or in a cavity which specifically binds to, and is complementarywith, a particular spatial and polar organization of the other.Exemplary binding pairs are set forth in Table 2.

TABLE 2 Representative Specific Binding Pairs antigen antibody biotinavidin (or streptavidin or anti-biotin) IgG* protein A or protein G drugdrug receptor folate folate binding protein toxin toxin receptorcarbohydrate lectin or carbohydrate receptor peptide peptide receptorprotein protein receptor enzyme substrate enzyme DNA (RNA) cDNA (cRNA)†hormone hormone receptor ion chelator *IgG is an immunoglobulin †cDNAand cRNA are the complementary strands used for hybridization

In an exemplary embodiment, the present compounds of the invention arecovalently bonded to a solid support. The solid support may be attachedto the compound either through the A, L or B moiety, or through areactive group, if present, or through a carrier molecule, if present.Even if a reactive group and/or a carrier molecule are present, thesolid support may be attached through the A, L or B moiety. In exemplaryembodiment, at least one member selected from A, L or B comprises asolid support. Preferably, at least one of A or B comprises a solidsupport, wherein at least one of R³, R⁴, R⁶ or R⁷ is a solid support oris attached to a solid support. In another aspect, L comprises a solidsupport; typically the E moiety is attached to a solid support.

A solid support suitable for use in the present invention is typicallysubstantially insoluble in liquid phases. Solid supports of the currentinvention are not limited to a specific type of support. Rather, a largenumber of supports are available and are known to one of ordinary skillin the art. Thus, useful solid supports include solid and semi-solidmatrixes, such as aerogels and hydrogels, resins, beads, biochips(including thin film coated biochips), microfluidic chip, a siliconchip, multi-well plates (also referred to as microtitre plates ormicroplates), membranes, conducting and nonconducting metals, glass(including microscope slides) and magnetic supports. More specificexamples of useful solid supports include silica gels, polymericmembranes, particles, derivatized plastic films, glass beads, cotton,plastic beads, alumina gels, polysaccharides such as Sepharose,poly(acrylate), polystyrene, poly(acrylamide), polyol, agarose, agar,cellulose, dextran, starch, FICOLL, heparin, glycogen, amylopectin,mannan, inulin, nitrocellulose, diazocellulose, polyvinylchloride,polypropylene, polyethylene (including poly(ethylene glycol)), nylon,latex bead, magnetic bead, paramagnetic bead, superparamagnetic bead,starch and the like.

In some embodiments, the solid support may include a solid supportreactive functional group, including, but not limited to, hydroxyl,carboxyl, amino, thiol, aldehyde, halogen, nitro, cyano, amido, urea,carbonate, carbamate, isocyanate, sulfone, sulfonate, sulfonamide,sulfoxide, etc., for attaching the compounds of the invention. Usefulreactive groups are disclosed above and are equally applicable to thesolid support reactive functional groups herein.

A suitable solid phase support can be selected on the basis of desiredend use and suitability for various synthetic protocols. For example,where amide bond formation is desirable to attach the compounds of theinvention to the solid support, resins generally useful in peptidesynthesis may be employed, such as polystyrene (e.g., PAM-resin obtainedfrom Bachem Inc., Peninsula Laboratories, etc.), POLYHIPE™ resin(obtained from Aminotech, Canada), polyamide resin (obtained fromPeninsula Laboratories), polystyrene resin grafted with polyethyleneglycol (TentaGel™, Rapp Polymere, Tubingen, Germany),polydimethyl-acrylamide resin (available from Milligen/Biosearch,California), or PEGA beads (obtained from Polymer Laboratories).

Preparation of Conjugates

Conjugates of components (carrier molecules or solid supports), e.g.,drugs, peptides, toxins, nucleotides, phospholipids and other organicmolecules are prepared by organic synthesis methods using the reactivereporter molecules of the invention, are generally prepared by meanswell recognized in the art (Haugland, MOLECULAR PROBES HANDBOOK, supra,(2002)). Preferably, conjugation to form a covalent bond consists ofsimply mixing the reactive compounds of the present invention in asuitable solvent in which both the reactive compound and the substanceto be conjugated are soluble. The reaction preferably proceedsspontaneously without added reagents at room temperature or below. Forthose reactive compounds that are photoactivated, conjugation isfacilitated by illumination of the reaction mixture to activate thereactive compound. Chemical modification of water-insoluble substances,so that a desired compound-conjugate may be prepared, is preferablyperformed in an aprotic solvent such as dimethylformamide,dimethylsulfoxide, acetone, ethyl acetate, toluene, or chloroform.Similar modification of water-soluble materials is readily accomplishedthrough the use of the instant reactive compounds to make them morereadily soluble in organic solvents.

Preparation of peptide or protein conjugates typically comprises firstdissolving the protein to be conjugated in aqueous buffer at about0.1-10 mg/mL at room temperature or below. Bicarbonate buffers (pH about8.3) are especially suitable for reaction with succinimidyl esters,phosphate buffers (pH about 7.2-8) for reaction with thiol-reactivefunctional groups and carbonate or borate buffers (pH about 9) forreaction with isothiocyanates and dichlorotriazines. The appropriatereactive compound is then dissolved in an aprotic solvent (usually DMSOor DMF) in an amount sufficient to give a suitable degree of labelingwhen added to a solution of the protein to be conjugated. Theappropriate amount of compound for any protein or other component isconveniently predetermined by experimentation in which variable amountsof the compound are added to the protein, the conjugate ischromatographically purified to separate unconjugated compound and thecompound-protein conjugate is tested in its desired application.

Following addition of the reactive compound to the component solution,the mixture is incubated for a suitable period (typically about 1 hourat room temperature to several hours on ice), the excess compound isremoved by gel filtration, dialysis, HPLC, adsorption on an ion exchangeor hydrophobic polymer or other suitable means. The compound-conjugateis used in solution or lyophilized. In this way, suitable conjugates canbe prepared from antibodies, antibody fragments, avidins, lectins,enzymes, proteins A and G, cellular proteins, albumins, histones, growthfactors, hormones, and other proteins.

Conjugates of polymers, including biopolymers and other higher molecularweight polymers are typically prepared by means well recognized in theart (for example, Brinkley et al., Bioconjugate Chem., 3: 2 (1992)). Inthese embodiments, a single type of reactive site may be available, asis typical for polysaccharides) or multiple types of reactive sites(e.g. amines, thiols, alcohols, phenols) may be available, as is typicalfor proteins. Selectivity of labeling is best obtained by selection ofan appropriate reactive dye. For example, modification of thiols with athiol-selective reagent such as a haloacetamide or maleimide, ormodification of amines with an amine-reactive reagent such as anactivated ester, acyl azide, isothiocyanate or3,5-dichloro-2,4,6-triazine. Partial selectivity can also be obtained bycareful control of the reaction conditions.

When modifying polymers with the compounds, an excess of compound istypically used, relative to the expected degree of compoundsubstitution. Any residual, unreacted compound or a compound hydrolysisproduct is typically removed by dialysis, chromatography orprecipitation. Presence of residual, unconjugated dye can be detected bythin layer chromatography using a solvent that elutes the dye away fromits conjugate. In all cases it is usually preferred that the reagents bekept as concentrated as practical so as to obtain adequate rates ofconjugation.

In an exemplary embodiment, the conjugate of the invention is associatedwith an additional substance, that binds either to the reporter moleculeor the conjugated substance (carrier molecule or solid support) throughnoncovalent interaction. In another exemplary embodiment, the additionalsubstance is an antibody, an enzyme, a hapten, a lectin, a receptor, anoligonucleotide, a nucleic acid, a liposome, or a polymer. Theadditional substance is optionally used to probe for the location of thedye-conjugate, for example, as a means of enhancing the signal of thedye-conjugate.

Nucleic Acid Reporter Molecules

The above described unsymmetrical cyanine monomer moieties and linkerscan be combined in numerous ways resulting in the nucleic acid reportermolecules of the present invention that are hetero- or homodimers.Typically, the monomer moieties form dimers via the pyridinium orquinolinium moieties wherein the linker is covalently bonded to thepyridinium or quinolinium moieties of both monomer moieties. In thisway, the present unsymmetrical cyanine dimer compounds have the generalformula, A-L-B or A-L*-B. More specifically, the present nucleic acidreporter molecules have the following formula:

wherein X, R³, R⁴, K and j are as defined above. The monomer moietiesmay be the same or different resulting in homo- or heterodimer compounds

Selected nucleic reporter molecules of the present invention include:

wherein X is S, the monomer moieties are the same and are according toFormula (III)(a) and the linker is according to Formula (II)(i),

Compound 26, wherein X is O and the monomer moieties are differentforming a heterodimer wherein one monomer is according to Formula(III)(b), the second monomer is according to Formula (III)(e) and thelinker is according to Formula (IV)(iv),

Compound 27, wherein X is O and the monomers are according to Formula(III)(a) and the linker is according to Formula (II)(f),

Compound 28, wherein X is S and the monomers are according to Formula(III)(a) and the linker is according to Formula (I),

wherein X is O,

Compound 11 wherein X is S, the monomer moieties are the same and areaccording to Formula (III)(b) and the linker is according to Formula(II)(j),

wherein the monomer moieties are the same and are according to Formula(III)(e) and the linker is according to Formula (II)(i),

wherein the monomer moieties are the same and are according to Formula(III)(b) and the linker is according to Formula (II)(i), and

wherein K

represents a pyridinium moiety (Formula (III)(a)) for one of the cyaninemonomers and a quinolinium moiety (Formula (III)(b)) for the othercyanine monomer. The linker is according to Formula (II)(j).

Presently preferred for detection of intracellular RNA are compounds 6and 19. Presently preferred compounds for the detection of extracellularRNA are compounds 11, 20 and 23.

B. Methods of Use

The present nucleic acid reporter molecules may be utilized withoutlimit for the fluorescent detection of nucleic acid polymers in a testsample. The methods for the detection of single, double, triple orquadruple stranded DNA and RNA or a combination thereof comprisescontacting a sample with a staining solution to prepare a labelingmixture, incubating the sample with the staining solution for asufficient amount of time for the present reporter molecules to complexwith the nucleic acid, illuminating the sample with an appropriatewavelength and observing the illuminated labeling mixture whereby thenucleic acid polymer is detected.

The staining solution is typically prepared by dissolving a presentnucleic acid reporter molecule in an aqueous solvent such as water, abuffer solution, such as phosphate buffered saline, or an organicsolvent such as dimethylsulfoxide (DMSO), dimethylformamide (DMF),methanol, ethanol or acetonitrile. Typically, the present nucleicreporter molecules are first dissolved in an organic solvent such asDMSO as a stock solution. The stock solution is then diluted to aneffective working concentration in an aqueous solution optionallycomprising appropriate buffering components. An effective workingconcentration of the present compounds is the amount sufficient to givea detectable fluorescent response when complexed with nucleic acidpolymers. Typically, the effective amount is about 100 nm to 100 μM.Most preferred is about 600 nm to 10 μM. It is generally understood thatthe specific amount of the nucleic acid reporter molecules present in astaining solution is determined by the physical nature of the sample andthe nature of the analysis being performed.

The sample may be combined with the staining solution by any means thatfacilitates contact between the nucleic acid reporter molecules and thenucleic acid. The contact can occur through simple mixing, as in thecase where the sample is a solution. The present reporter molecules maybe added to the nucleic acid solution directly or may contact thesolution on an inert matrix such as a blot or gel, a testing strip, amicroarray, or any other solid or semi-solid surface, for example whereonly a simple and visible demonstration of the presence of nucleic acidsis desired. Any inert matrix used to separate the sample can be used todetect the presence of nucleic acids by observing the fluorescentresponse on the inert matrix. While the present reporter molecules haveshown an ability to permeate cellular membranes rapidly and completelyupon addition of the staining solution, any other technique that issuitable for transporting the reporter molecules across cell membraneswith minimal disruption of the viability of the cell and integrity ofcell membranes is also a valid method of combining the sample with thepresent reporter molecules for detection of intracellular nucleic acid.Examples of suitable processes include action of chemical agents such asdetergents, enzymes or adenosine triphosphate; receptor- or transportprotein-mediated uptake; pore-forming proteins; microinjection;electroporation; hypoosmotic shock; or minimal physical disruption suchas scrape loading or bombardment with solid particles coated with or inthe presence of the present reporter molecules.

The sample is incubated in the presence of the nucleic acid reportermolecules for a time sufficient to form the fluorescent nucleicacid-reporter molecule complex. Detectable fluorescence in a solution ofnucleic acids is essentially instantaneous. Detectable fluorescencewithin cell membranes requires the permeation of the dye into the cell.In general, visibly detectable fluorescence can be obtained in a widevariety of cells with embodiments of the present invention within about10-30 minutes after combination with the sample, commonly within about10-20 minutes. While permeation and fluorescence is rapid for allreporter molecules comprising an aromatic substituent on the pyridiniumor quinolinium moiety of the cyanine monomer moiety, it is readilyapparent to one skilled in the art that the time necessary forsufficient permeation of the dye, or sufficient formation of thefluorescent nucleic acid complex, is dependent upon the physical andchemical nature of the individual sample and the sample medium.

To facilitate the detection of the nucleic acid-reporter moleculecomplex, the excitation or emission properties of the fluorescentcomplex are utilized. For example, the sample is excited by a lightsource capable of producing light at or near the wavelength of maximumabsorption of the fluorescent complex, such as an ultraviolet or visiblelamp, an arc lamp, a laser, or even sunlight. Preferably the fluorescentcomplex is excited at a wavelength equal to or greater than about 300nm, more preferably equal to or greater than about 340 nm. Thefluorescence of the complex is detected qualitatively or quantitativelyby detection of the resultant light emission at a wavelength of greaterthan about 400 nm, more preferably greater than about 450 nm, mostpreferred greater than 480 nm. The emission is detected by means thatinclude visible inspection, photographic film, or the use of currentinstrumentation such as fluorometers, quantum counters, plate readers,epifluorescence microscopes and flow cytometers or by means foramplifying the signal such as a photomultiplier.

The present invention also provides specific compounds and methods fordetecting RNA in the presence of DNA, wherein the method comprises thefollowing steps:

-   -   a) combining a present nucleic acid reporter molecule with a        sample wherein the reporter molecule is capable of producing a        fluorescent intensity signal that is greater on RNA than on DNA        to prepare a labeling mixture;    -   b) incubating the labeling mixture for a sufficient amount of        time for said nucleic acid reporter molecule to associate with        RNA in the sample; and,    -   c) illuminating the labeling mixture with an appropriate        wavelength; and    -   d) observing the illuminated labeling mixture whereby the RNA is        detected in the presence of DNA.

The present nucleic acid reporter molecules that are capable ofproducing a fluorescent intensity signal that is greater on RNA than onDNA are determined empirically, See Examples 35 and 36 and Table 3.However, we have found that cyanine monomer moieties that comprise amonomethine bridge and a rigid linker according to Formula (II)(b),(II)(i), (II)(ia), II(ib) and (II)(j) produce nucleic acid reportermolecules that have a greater fluorescent intensity signal on RNA thanon DNA. It is not intended that only this class of present nucleic acidreporter molecules are capable of having a greater fluorescent intensitysignal on RNA than DNA as other classes within the scope of theinvention may also be used to detect RNA in the presence of DNA. Withoutwishing to be bound by a theory, it appears that the reporter moleculescapable of fluorescing brighter on RNA than on DNA preferentiallycomplex with RNA. This may be due to the size of the helical groove ofRNA for which rigid linkers according to Formula (II)(a), (II)(b),(II)(i) and (II)(j) preferentially form a RNA-reporter molecule complex.Thus, preferred compounds for the detection of RNA include Compound 6,11, 19, 20 and 23.

The foregoing methods having been described it is understood that themany and varied compounds of the present invention can be utilized withthe many methods. The compounds not being limited to just those that arespecifically disclosed.

In an exemplary embodiment the present methods employ a nucleic acidreporter molecule that comprises a first nucleic acid complexing monomermoiety, a second nucleic acid complexing monomer moiety and a linkerthat comprises at least one aromatic, heteroaromatic, cyclic orheterocyclic moiety comprising 3-20 non-hydrogen atoms selected from thegroup consisting of O, N, S, P and C, wherein the first nucleic acidcomplexing monomer moiety is covalently attached to the linker; and thesecond nucleic acid complexing monomer moiety is covalently attached tothe linker. In one aspect, the first and the second first nucleic acidcomplexing monomer moieties are the same wherein the present compound isa homodimer. In another aspect, the first and the second first nucleicacid complexing monomer moieties are different wherein the presentcompound is a heterodimer.

In an exemplary embodiment of the methods the nucleic acid complexingmonomer moiety is an unsymmetrical cyanine monomer moiety, wherein thepresent nucleic acid reporter molecule comprises a first unsymmetricalcyanine monomer moiety, a second unsymmetrical cyanine monomer moiety,and a linker that comprises at least one aromatic, heteroaromatic,cyclic or heterocyclic moiety comprising 3-20 non-hydrogen atomsselected from the group consisting of O, N, S, P and C, wherein thefirst unsymmetrical cyanine monomer moiety is covalently attached to thelinker; and the second unsymmetrical cyanine monomer moiety iscovalently attached to the linker. In one aspect the first and thesecond unsymmetrical cyanine monomer moieties are the same, wherein thepresent compound is cyanine homodimer. In another aspect, the first andthe second unsymmetrical cyanine monomer moieties are different, whereinthe present compound is a cyanine heterodimer.

In an exemplary embodiment the methods employ a present nucleic acidreporter molecule is according to the formula:

-   -   wherein each R³ is independently hydrogen, unsubstituted C₁-C₆        alkyl, substituted C₁-C₆ alkyl, unsubstituted C₁-C₆ alkoxy,        substituted C₁-C₆ alkoxy, unsubstituted fused benzene,        substituted fused benzene, unsubstituted trifluoromethyl,        substituted trifluoromethyl, unsubstituted halogen, substituted        halogen, unsubstituted reactive group, substituted reactive        group, unsubstituted carrier molecule, substituted carrier        molecule, unsubstituted solid support or substituted solid        support;    -   each R⁴ is independently a unsubstituted C₁-C₆ alkyl,        substituted C₁-C₆ alkyl, unsubstituted reactive group,        substituted reactive group, unsubstituted carrier molecule,        substituted carrier molecule, unsubstituted solid support or        substituted solid support;    -   X is O, S, or CR⁶R⁷ wherein each R⁶ and R⁷ are independently a        unsubstituted C₁-C₆ alkyl, substituted C₁-C₆ alkyl,        unsubstituted reactive group, substituted reactive group,        unsubstituted carrier molecule, substituted carrier molecule,        unsubstituted solid support, substituted solid support or R⁶ and        R⁷ taken together form a 5- or 6-membered saturated ring;    -   j is 0, 1, or 2;    -   linker is a series of stable covalent bonds comprising 1-30        nonhydrogen atoms selected from the group consisting of C, N, O,        S and P; and,    -   K is substituted pyridinium, unsubstituted pyridinium,        substituted quinolinium, or unsubstituted quinolinium.

Thus, in one embodiment, the unsymmetrical cyanine monomer is accordingto the formula:

wherein the linker is covalently attached to the K moiety of the cyaninemonomer.

Thus, in a particular embodiment, when the linker does not comprises atleast one aromatic, heteroaromatic, cyclic or heterocyclic moiety, themethods use Compound 1, Compound 2, Compound 3, Compound 5, Compound 12,Compound 13, Compound 17, and Compound 22. These compounds are cellpermeable and in a particularly useful embodiment Compound 1 is used toselectively detect mitochondrial DNA, See Example 43.

In another exemplary embodiment of the methods, a nucleic acid reportermolecule comprising a first unsymmetrical cyanine monomer moiety, asecond unsymmetrical cyanine monomer moiety, and a linker having formula(I)—(Y)_(r)—(CH₂)_(m)-T_(q)-(CH₂)_(n)-E-(CH₂)_(n)-T_(q)-(CH₂)_(m)—(Y)_(r)—is employed. Y is a linear or branched moiety comprising 1-20non-hydrogen atoms selected from the group consisting of C, N, O, P andS; T is a unsubstituted heteroatom or a substituted heteroatom; E is anaromatic, heteroaromatic, cyclic or heterocyclic moiety comprising 3-20non-hydrogen atoms selected from the group consisting of O, N, S, P andC; r is independently 0 or 1; m is an integer of 0-6; n is independently0 or 1; q is independently 0 or 1; and wherein the first unsymmetricalcyanine monomer moiety is covalently attached to the linker; and thesecond unsymmetrical cyanine monomer moiety is covalently attached tothe linker.

In one aspect, E of the linker is

wherein R² is hydrogen, amine, substituted amine, substituted C₁-C₆alkyl, unsubstituted C₁-C₆ alkyl, unsubstituted reactive group,substituted reactive group, unsubstituted carrier molecule, substitutedcarrier molecule, unsubstituted solid support, or substituted solidsupport. In a further aspect T of the linker, when present, is —NR², —N═or S wherein the R² is hydrogen, amine, substituted amine, substitutedC₁-C₆ alkyl, unsubstituted C₁-C₆ alkyl, unsubstituted reactive group,substituted reactive group, unsubstituted carrier molecule, substitutedcarrier molecule, unsubstituted solid support, or substituted solidsupport.

In an exemplary embodiment, the nucleic aid reporter molecule of thepresent methods comprise a reactive group, solid support and carriermolecule wherein these substituents independently comprise a linker thatis a single covalent bond, or a covalent linkage that is linear orbranched, cyclic or heterocyclic, saturated or unsaturated, having 1-20nonhydrogen atoms selected from the group consisting of C, N, P, O andS; and are composed of any combination of ether, thioether, amine,ester, carboxamide, sulfonamide, hydrazide bonds and aromatic orheteroaromatic bonds.

In an exemplary embodiment, the reactive group is selected from thegroup consisting of an acrylamide, an activated ester of a carboxylicacid, a carboxylic ester, an acyl azide, an acyl nitrile, an aldehyde,an alkyl halide, an anhydride, an aniline, an amine, an aryl halide, anazide, an aziridine, a boronate, a diazoalkane, a haloacetamide, ahaloalkyl, a halotriazine, a hydrazine, an imido ester, an isocyanate,an isothiocyanate, a maleimide, a phosphoramidite, a reactive platinumcomplex, a silyl halide, a sulfonyl halide, a thiol and aphotoactivatable group. In a further aspect, the reactive group isselected from the group consisting of carboxylic acid, succinimidylester of a carboxylic acid, hydrazide, amine and a maleimide.

In an exemplary embodiment the carrier molecule is selected from thegroup consisting of an amino acid, a peptide, a protein, apolysaccharide, a nucleoside, a nucleotide, an oligonucleotide, anucleic acid polymer, a hapten, a psoralen, a drug, a hormone, a lipid,a lipid assembly, a synthetic polymer, a polymeric microparticle, abiological cell or a virus. In a further aspect, the carrier molecule isselected from the group consisting of an antibody or fragment thereof,an avidin or streptavidin, a biotin, a blood component protein, adextran, an enzyme, an enzyme inhibitor, a hormone, an IgG bindingprotein, a fluorescent protein, a growth factor, a lectin, alipopolysaccharide, a microorganism, a metal binding protein, a metalchelating moiety, a non-biological microparticle, a peptide toxin, aphosphotidylserine-binding protein, a structural protein, asmall-molecule drug, or a tyramide.

In an exemplary embodiment, the solid support is selected from the groupconsisting of a microfluidic chip, a silicon chip, a microscope slide, amicroplate well, silica gels, polymeric membranes, particles,derivatized plastic films, glass beads, cotton, plastic beads, aluminagels, polysaccharides, polyvinylchloride, polypropylene, polyethylene,nylon, latex bead, magnetic bead, paramagnetic bead, andsuperparamagnetic bead. In a further aspect, the solid support isselected from the group consisting of Sepharose, poly(acrylate),polystyrene, poly(acrylamide), polyol, agarose, agar, cellulose,dextran, starch, FICOLL, heparin, glycogen, amylopectin, mannan, inulin,nitrocellulose, diazocellulose and starch.

In an exemplary embodiment of the present methods, the linker of thenucleic acid reporter molecule is

Wherein each R⁸, R⁹, R¹⁰, R¹¹ and R¹² are independently a —NR², —N═ or Swherein R² is hydrogen, amine, substituted amine, substituted C₁-C₆alkyl, unsubstituted C₁-C₆ alkyl, unsubstituted reactive group,substituted reactive group, unsubstituted carrier molecule, substitutedcarrier molecule, unsubstituted solid support, or substituted solidsupport; each Z is independently —(CH₂)_(g)— wherein g is 0 or 1,wherein the dashed line is attached directly to the K moiety of theunsymmetrical cyanine monomer. In one aspect R⁸ is S. In a furtheraspect, R¹² is —NH or —N═.

Particularly preferred for the detection of nucleic acid are reportermolecules wherein the linker is

In a particularly preferred embodiment, the nucleic acid reportermolecules are employed to detect RNA in the presence of DNA wherein thenucleic acid reporter molecule has a RNA/DNA ratio of fluorescenceenhancement greater than about one. In this instance the nucleic acidreporter molecule comprises a first monomethine unsymmetrical cyaninemonomer moiety, a second monomethine unsymmetrical cyanine monomermoiety and a linker that is

wherein R⁸ is a substituted heteroatom or an unsubstituted heteroatom;R⁹ is a substituted heteroatom or an unsubstituted heteroatom; R¹² is asubstituted heteroatom or an unsubstituted heteroatom; each Z is—(CH₂)_(g) wherein g is 0 or 1; and the first unsymmetrical cyaninemonomer moiety is covalently attached to the linker; and the secondunsymmetrical cyanine monomer moiety is covalently attached to thelinker. In a further embodiment, R⁸, R⁹ and R¹² are —NR², —N═ or Swherein R² is hydrogen, amine, substituted amine, substituted C₁-C₆alkyl, unsubstituted C₁-C₆ alkyl, unsubstituted reactive group,substituted reactive group, unsubstituted carrier molecule, substitutedcarrier molecule, unsubstituted solid support, or substituted solidsupport. In one aspect, Compound 6 is preferred for the detection ofintracellular RNA.

In an exemplary embodiment, the monomethine unsymmetrical cyaninemonomer moiety has the general formula:

wherein R³, R4, X and K are as defined above.

Sample Preparation

The end user will determine the choice of the sample and the way inwhich the sample is prepared but the sample is typically prepared usingmethods well known in the art for isolating nucleic acid for in vitroand solution based assay detection or well know methods for live cell orfixed cells for intracellular and/or in vivo detection of nucleic acids.The sample includes, without limitation, any biological derived materialthat is thought to contain a nucleic acid polymer. Alternatively,samples also include material that nucleic acid polymers have been addedto such as a PCR reaction mixture, a polymer gel such as agarose orpolyacrylamide gels or a microfluidic assay system. In another aspect ofthe invention, the sample can also include a buffer solution thatcontains nucleic acid polymers to determine the present reportermolecules that are ideal under different assay conditions or todetermine the present reporter molecules that are preferential RNAreporters.

The sample can be a biological fluid such as whole blood, plasma, serum,nasal secretions, sputum, saliva, urine, sweat, transdermal exudates,cerebrospinal fluid, or the like. Biological fluids also include tissueand cell culture medium wherein an analyte of interest has been secretedinto the medium. Alternatively, the sample may be whole organs, tissueor cells from the animal. Examples of sources of such samples includemuscle, eye, skin, gonads, lymph nodes, heart, brain, lung, liver,kidney, spleen, thymus, pancreas, solid tumors, macrophages, mammaryglands, mesothelium, and the like. Cells include without limitationprokaryotic cells such as bacteria, yeast, fungi, mycobacteria andmycoplasma, and eukaryotic cells such as nucleated plant and animalcells that include primary cultures and immortalized cell lines.Typically prokaryotic cells include E. coli and S. aureus. Eukaryoticcells include without limitation ovary cells, epithelial cells,circulating immune cells, β cells, hepatocytes, and neurons.

The nucleic acid may be either natural (biological in origin) orsynthetic (prepared artificially). The nucleic acid may be present asnucleic acid fragments, oligonucleotides, or nucleic acid polymers. Thenucleic acid may be present in a condensed phase, such as a chromosome.The presence of the nucleic acid in the sample may be due to asuccessful or unsuccessful experimental methodology, undesirablecontamination, or a disease state. Nucleic acid may be present in all,or only part, of a sample, and the presence of nucleic acids may be usedto distinguish between individual samples, or to differentiate a portionor region within a single sample.

The nucleic acid may be enclosed in a biological structure, for examplecontained within a viral particle, an organelle, or within a cell. Thenucleic acids enclosed in biological structures may be obtained from awide variety of environments, including cultured cells, organisms ortissues, unfiltered or separated biological fluids such as urine,cerebrospinal fluid, blood, lymph fluids, tissue homogenate, mucous,saliva, stool, or physiological secretions or environmental samples suchas soil, water and air. The nucleic acid may be endogenous or introducedas foreign material, such as by infection or by transfection. Thepresent nucleic acid reporter molecules can also be used for stainingnucleic acids in a cell or cells that is fixed and treated with routinehistochemical or cytochemical procedures (See, Examples 29-31).

Alternatively, the nucleic acid is not enclosed within a biologicalstructure, but is present as a sample solution. The sample solution canvary from one of purified nucleic acids to crude mixtures such as cellextracts, biological fluids and environmental samples. In some cases itis desirable to separate the nucleic acids from a mixture ofbiomolecules or fluids in the solution prior to combination with thepresent reporter molecules. Numerous, well known, techniques exist forseparation and purification of nucleic acids from generally crudemixtures with other proteins or other biological molecules. Theseinclude such means as electrophoretic techniques and chromatographictechniques using a variety of supports.

Illumination

The sample containing a nucleic acid-reporter molecule complex isilluminated with a wavelength of light selected to give a detectableoptical response, and observed with a means for detecting the opticalresponse. Equipment that is useful for illuminating the presentcompounds and compositions of the invention includes, but is not limitedto, hand-held ultraviolet lamps, mercury arc lamps, xenon lamps, lasersand laser diodes. These illumination sources are optically integratedinto laser scanners, fluorescence microplate readers or standard ormicrofluorometers.

The optical response is optionally detected by visual inspection, or byuse of any of the following devices: CCD camera, video camera,photographic film, laser-scanning devices, fluorometers, photodiodes,quantum counters, epifluorescence microscopes, scanning microscopes,flow cytometers, fluorescence microplate readers, or by means foramplifying the signal such as photomultiplier tubes. Where the sample isexamined using a flow cytometer, examination of the sample optionallyincludes sorting portions of the sample according to their fluorescenceresponse.

The wavelengths of the excitation and emission bands of the nucleic acidreporter molecules vary with reporter molecule composition to encompassa wide range of illumination and detection bands. This allows theselection of individual reporter molecules for use with a specificexcitation source or detection filter. In particular, present reportermolecules can be selected that possess excellent correspondence of theirexcitation band with the 488 nm band of the commonly used argon laser oremission bands which are coincident with preexisting filters

Kits of the Invention

Suitable kits for forming a nucleic acid-reporter molecule complex anddetecting the nucleic acid also form part of the invention. Such kitscan be prepared from readily available materials and reagents and cancome in a variety of embodiments. The contents of the kit will depend onthe design of the assay protocol or reagent for detection ormeasurement. All kits will contain instructions, appropriate reagents,and present nucleic acid reporter molecules. Typically, instructionsinclude a tangible expression describing the reagent concentration or atleast one assay method parameter such as the relative amounts of reagentand sample to be added together, maintenance time periods forreagent/sample admixtures, temperature, buffer conditions and the liketo allow the user to carry out any one of the methods or preparationsdescribed above.

Thus in one aspect of the invention, a kit for detecting nucleic acid ina sample comprises

-   -   a) a present nucleic acid reporter molecule.

In a further aspect the kit comprises instructions for detecting thenucleic acid, presently preferred are instructions for detecting RNA. Inanother aspect the kit may contain one or more of the following; samplepreparation reagents, a buffering agent, an organic solvent or anadditional nucleic acid reporter molecule.

The additional nucleic acid reporter molecule is typically one thatdistinguishes a different aspect of the sample of nucleic acid from thepresent reporter molecule.

The foregoing kits having been described it is understood that the manyand varied compounds of the present invention can be utilized with themany kits. The compounds not being limited to just those that arespecifically disclosed.

In an exemplary embodiment the present kits employ a nucleic acidreporter molecule that comprises a first nucleic acid complexing monomermoiety, a second nucleic acid complexing monomer moiety and a linkerthat comprises at least one aromatic, heteroaromatic, cyclic orheterocyclic moiety comprising 3-20 non-hydrogen atoms selected from thegroup consisting of O, N, S, P and C, wherein the first nucleic acidcomplexing monomer moiety is covalently attached to the linker; and thesecond nucleic acid complexing monomer moiety is covalently attached tothe linker. In one aspect, the first and the second first nucleic acidcomplexing monomer moieties are the same wherein the present compound isa homodimer. In another aspect, the first and the second first nucleicacid complexing monomer moieties are different wherein the presentcompound is a heterodimer.

In an exemplary embodiment of the kits the nucleic acid complexingmonomer moiety is an unsymmetrical cyanine monomer moiety, wherein thepresent nucleic acid reporter molecule comprises a first unsymmetricalcyanine monomer moiety, a second unsymmetrical cyanine monomer moiety,and a linker that comprises at least one aromatic, heteroaromatic,cyclic or heterocyclic moiety comprising 3-20 non-hydrogen atomsselected from the group consisting of O, N, S, P and C, wherein thefirst unsymmetrical cyanine monomer moiety is covalently attached to thelinker; and the second unsymmetrical cyanine monomer moiety iscovalently attached to the linker. In one aspect the first and thesecond unsymmetrical cyanine monomer moieties are the same, wherein thepresent compound is cyanine homodimer. In another aspect, the first andthe second unsymmetrical cyanine monomer moieties are different, whereinthe present compound is a cyanine heterodimer.

In an exemplary embodiment the kits employ a present nucleic acidreporter molecule is according to the formula:

-   -   wherein each R³ is independently hydrogen, unsubstituted C₁-C₆        alkyl, substituted C₁-C₆ alkyl, unsubstituted C₁-C₆ alkoxy,        substituted C₁-C₆ alkoxy, unsubstituted fused benzene,        substituted fused benzene, unsubstituted trifluoromethyl,        substituted trifluoromethyl, unsubstituted halogen, substituted        halogen, unsubstituted reactive group, substituted reactive        group, unsubstituted carrier molecule, substituted carrier        molecule, unsubstituted solid support or substituted solid        support;    -   each R⁴ is independently a unsubstituted C₁-C₆ alkyl,        substituted C₁-C₆ alkyl, unsubstituted reactive group,        substituted reactive group, unsubstituted carrier molecule,        substituted carrier molecule, unsubstituted solid support or        substituted solid support;    -   X is O, S, or CR⁶R⁷ wherein each R⁶ and R⁷ are independently a        unsubstituted C₁-C₆ alkyl, substituted C₁-C₆ alkyl,        unsubstituted reactive group, substituted reactive group,        unsubstituted carrier molecule, substituted carrier molecule,        unsubstituted solid support, substituted solid support or R⁶ and        R⁷ taken together form a 5- or 6-membered saturated ring;    -   j is 0, 1, or 2;    -   linker is a series of stable covalent bonds comprising 1-30        nonhydrogen atoms selected from the group consisting of C, N, O,        S and P; and,    -   K is substituted pyridinium, unsubstituted pyridinium,        substituted quinolinium, or unsubstituted quinolinium.

Thus, in one embodiment, the unsymmetrical cyanine monomer is accordingto the formula:

wherein the linker is covalently attached to the K moiety of the cyaninemonomer.

Thus, in a particular embodiment, when the linker does not comprises atleast one aromatic, heteroaromatic, cyclic or heterocyclic moiety, thekits use Compound 1, Compound 2, Compound 3, Compound 5, Compound 12,Compound 13, Compound 17, and Compound 22. These compounds are cellpermeable and in a particularly useful embodiment Compound 1 is used toselectively detect mitochondrial DNA, See Example 43.

In another exemplary embodiment of the kits, a nucleic acid reportermolecule comprising a first unsymmetrical cyanine monomer moiety, asecond unsymmetrical cyanine monomer moiety, and a linker having formula(I)—(Y)_(r)—(CH₂)_(m)-T_(q)-(CH₂)_(n)-E-(CH₂)_(n)-T_(q)-(CH₂)_(m)—(Y)_(r)—is employed. Y is a linear or branched moiety comprising 1-20non-hydrogen atoms selected from the group consisting of C, N, O, P andS; T is a unsubstituted heteroatom or a substituted heteroatom; E is anaromatic, heteroaromatic, cyclic or heterocyclic moiety comprising 3-20non-hydrogen atoms selected from the group consisting of O, N, S, P andC; r is independently 0 or 1; m is an integer of 0-6; n is independently0 or 1; q is independently 0 or 1; and wherein the first unsymmetricalcyanine monomer moiety is covalently attached to the linker; and thesecond unsymmetrical cyanine monomer moiety is covalently attached tothe linker.

In one aspect, E of the linker is

wherein R² is hydrogen, amine, substituted amine, substituted C₁-C₆alkyl, unsubstituted C₁-C₆ alkyl, unsubstituted reactive group,substituted reactive group, unsubstituted carrier molecule, substitutedcarrier molecule, unsubstituted solid support, or substituted solidsupport. In a further aspect T of the linker, when present, is —NR², —N═or S wherein the R² is hydrogen, amine, substituted amine, substitutedC₁-C₆ alkyl, unsubstituted C₁-C₆ alkyl, unsubstituted reactive group,substituted reactive group, unsubstituted carrier molecule, substitutedcarrier molecule, unsubstituted solid support, or substituted solidsupport.

In an exemplary embodiment, the nucleic aid reporter molecule of thepresent kits comprise a reactive group, solid support and carriermolecule wherein these substituents independently comprise a linker thatis a single covalent bond, or a covalent linkage that is linear orbranched, cyclic or heterocyclic, saturated or unsaturated, having 1-20nonhydrogen atoms selected from the group consisting of C, N, P, O andS; and are composed of any combination of ether, thioether, amine,ester, carboxamide, sulfonamide, hydrazide bonds and aromatic orheteroaromatic bonds.

In an exemplary embodiment, the reactive group is selected from thegroup consisting of an acrylamide, an activated ester of a carboxylicacid, a carboxylic ester, an acyl azide, an acyl nitrile, an aldehyde,an alkyl halide, an anhydride, an aniline, an amine, an aryl halide, anazide, an aziridine, a boronate, a diazoalkane, a haloacetamide, ahaloalkyl, a halotriazine, a hydrazine, an imido ester, an isocyanate,an isothiocyanate, a maleimide, a phosphoramidite, a reactive platinumcomplex, a silyl halide, a sulfonyl halide, a thiol and aphotoactivatable group. In a further aspect, the reactive group isselected from the group consisting of carboxylic acid, succinimidylester of a carboxylic acid, hydrazide, amine and a maleimide.

In an exemplary embodiment the carrier molecule is selected from thegroup consisting of an amino acid, a peptide, a protein, apolysaccharide, a nucleoside, a nucleotide, an oligonucleotide, anucleic acid polymer, a hapten, a psoralen, a drug, a hormone, a lipid,a lipid assembly, a synthetic polymer, a polymeric microparticle, abiological cell or a virus. In a further aspect, the carrier molecule isselected from the group consisting of an antibody or fragment thereof,an avidin or streptavidin, a biotin, a blood component protein, adextran, an enzyme, an enzyme inhibitor, a hormone, an IgG bindingprotein, a fluorescent protein, a growth factor, a lectin, alipopolysaccharide, a microorganism, a metal binding protein, a metalchelating moiety, a non-biological microparticle, a peptide toxin, aphosphotidylserine-binding protein, a structural protein, asmall-molecule drug, or a tyramide.

In an exemplary embodiment, the solid support is selected from the groupconsisting of a microfluidic chip, a silicon chip, a microscope slide, amicroplate well, silica gels, polymeric membranes, particles,derivatized plastic films, glass beads, cotton, plastic beads, aluminagels, polysaccharides, polyvinylchloride, polypropylene, polyethylene,nylon, latex bead, magnetic bead, paramagnetic bead, andsuperparamagnetic bead. In a further aspect, the solid support isselected from the group consisting of Sepharose, poly(acrylate),polystyrene, poly(acrylamide), polyol, agarose, agar, cellulose,dextran, starch, FICOLL, heparin, glycogen, amylopectin, mannan, inulin,nitrocellulose, diazocellulose and starch.

In an exemplary embodiment of the present kits, the linker of thenucleic acid reporter molecule is

Wherein each R⁸, R⁹, R¹⁰, R¹¹ and R¹² are independently a —NR², —N═ or Swherein R² is hydrogen, amine, substituted amine, substituted C₁-C₆alkyl, unsubstituted C₁-C₆ alkyl, unsubstituted reactive group,substituted reactive group, unsubstituted carrier molecule, substitutedcarrier molecule, unsubstituted solid support, or substituted solidsupport; each Z is independently —(CH₂)_(g)— wherein g is 0 or 1,wherein the dashed line is attached directly to the K moiety of theunsymmetrical cyanine monomer. In one aspect R⁸ is S. In a furtheraspect, R¹² is —NH or —N═.

Particularly preferred for the detection of nucleic acid is reportermolecules wherein the linker is

In a particularly preferred embodiment, the nucleic acid reportermolecules are employed to detect RNA in the presence of DNA wherein thenucleic acid reporter molecule has a RNA/DNA ratio of fluorescenceenhancement greater than about one. In this instance the nucleic acidreporter molecule comprises a first monomethine unsymmetrical cyaninemonomer moiety, a second monomethine unsymmetrical cyanine monomermoiety and a linker that is

wherein R⁸ is a substituted heteroatom or an unsubstituted heteroatom;R⁹ is a substituted heteroatom or an unsubstituted heteroatom; R¹² is asubstituted heteroatom or an unsubstituted heteroatom; each Z is—(CH₂)_(g) wherein g is 0 or 1; and the first unsymmetrical cyaninemonomer moiety is covalently attached to the linker; and the secondunsymmetrical cyanine monomer moiety is covalently attached to thelinker. In a further embodiment, R⁸, R⁹ and R¹² are —NR², —N═ or Swherein R² is hydrogen, amine, substituted amine, substituted C₁-C₆alkyl, unsubstituted C₁-C₆ alkyl, unsubstituted reactive group,substituted reactive group, unsubstituted carrier molecule, substitutedcarrier molecule, unsubstituted solid support, or substituted solidsupport. In an exemplary embodiment, Compound 6 is preferred for thedetection of intracellular RNA.

In an exemplary embodiment, the monomethine unsymmetrical cyaninemonomer moiety has the general formula:

wherein R³, R⁴, X and K are as defined above.

A detailed description of the invention having been provided above, thefollowing examples are given for the purpose of illustrating theinvention and shall not be construed as being a limitation on the scopeof the invention or claims.

EXAMPLES Example 1 Preparation of Compound 1

To 52 mg of2-chloro-4-(2,3-dihydro-3-methyl-(benzo-1,3-thiazol-2-yl)-methylidene-1-phenylquinoliniumiodide in 5 mL of methylene chloride, 19 mg of 1,9-nonanedithiol isadded followed by 20 μL of triethylamine. The mixture is stirred at roomtemperature overnight and 3.5 mL of ethyl acetate is then added toprecipitate out Compound 1.

Example 2 Preparation of Compound 2

A mixture of 150 mg of2-chloro-4-(2,3-dihydro-3-methyl-(benzo-1,3-thiazol-2-yl)-methylidene-1-phenylquinoliniumchloride, 32 mg of 4,7,10-trioxa-1,13-tridecanediamine, and 48 μL oftriethylamine is heated in 10 mL of dichloroethane at 40-50 C for 4hours. The solvent is evaporated and the residue is dissolved in 2 mL ofDMF and added to 0.43 g of sodium iodide in 10 mL of water. Compound 2is collected by filtration.

Example 3 Preparation of Compound 3

A mixture of 26 mg2-(3-succinimidyloxycarbonylethylthio)-4-(2,3-dihydro-3-methyl-(benzo-1,3-thiazol-2-yl)-methylidene-1-phenylquinoliniumiodide, 2.7 mg of 3,3′-diamino-n-methyldipropylamine, and 7 μL oftriethylamine is stirred in 1 mL of DMF at room temperature for 30minutes. At the end of the period, 2 mL of ethyl acetate is added toprecipitate Compound 3.

Example 4 Preparation of Compound 4

A mixture of 27 mg of2-(3-succinimidyloxycarbonylethylthio)-4-(2,3-dihydro-3-methyl-(benzo-1,3-thiazol-2-yl)-methylidene-1-phenylquinoliniumiodide, 3.9 mg of 1,4-bis(3-aminopropyl)piperazine and 7 μL oftriethylamine is stirred at room temperature for 30 minutes and 2 mL ofethyl acetate is then added to precipitate out Compound 4.

Example 5 Preparation of Compound 5

A mixture of 59 mg of2-chloro-4-(2,3-dihydro-3-methyl-(benzo-1,3-thiazol-2-yl)-methylidene-1-phenylquinoliniumiodide, 12 mg of N,N′-bis(2-mercaptoethyl)succinamide, and 17 μL oftriethylamine is stirred at room temperature for 30 minutes and Compound5 is collected by filtration.

Example 6 Preparation of Compound 6

A mixture of 537 mg of2-chloro-4-(2,3-dihydro-3-methyl-(benzo-1,3-thiazol-2-yl)-methylidene-1-phenylquinoliniumchloride, 40 mg of piperazine, and 0.13 mL of triethylamine is heated in10 mL of dichloroethane at 60 C for 4 hours. Compound 6 is collected byfiltration.

Example 7 Preparation of Compound 7

A mixture of 300 mg of2-chloro-4-(2,3-dihydro-3-methyl-(benzo-1,3-thiazol-2-yl)-methylidene-1-phenylquinoliniumchloride, 35 mg of dimercapto-1,3,4-thiadiazole, and 0.2 mL oftriethylamine is stirred in 10 mL of dichloroethane overnight andCompound 7 is collected by filtration.

Example 8 Preparation of Compound 8

A mixture of 300 mg of2-chloro-4-(2,3-dihydro-3-methyl-(benzo-1,3-thiazol-2-yl)-methylidene-1-phenylquinoliniumchloride, 26 mg of trans-1,4-diaminocyclohexane, and 0.2 mL oftriethylamine is heated in 10 mL of dichloroethane at 60 C overnight andCompound 8 is collected by filtration.

Example 9 Preparation of Compound 9

A mixture of 123 mg of2-chloro-4-(2,3-dihydro-3-methyl-(benzo-1,3-thiazol-2-yl)-methylidene-1-phenylquinoliniumchloride, 11 mg of 1,4-diaminopiperazine, and 0.12 mL of triethylamineis heated in 3 mL of DMF at 60 C for 5 hours and Compound 9 is collectedby filtration.

Example 10 Preparation of Compound 10

A mixture of 282 mg of4-chloro-2-(2,3-dihydro-3-methyl-(benzo-1,3-thiazol-2-yl)-methylidene-1-phenylquinoliniumchloride, 25 mg of 1,4-diaminopiperazine, and 0.25 mL of triethylamineis heated in 15 mL of DMF at 60 C for 2 hours and Compound 10 iscollected by filtration.

Example 11 Preparation of Compound 11

A mixture of 90 mg of2-chloro-4-(2,3-dihydro-3-methyl-(benzo-1,3-thiazol-2-yl)-methylidene-1-phenylpyridiniumchloride, 9 mg of piperazine, and 0.035 mL of triethylamine is heated in5 mL of DMF at 60 C for 2 hours and 15 mL of ethyl acetate is added andCompound 11 is collected by filtration.

Example 12 Preparation of Compound 12

A mixture of 0.2 g of 1,10-bis(4-methyl-quninolon-1-yl)decane, 0.8 mL oftrimethylsilytrifluoromethanesulfonate, 0.61 g of diisopropylethylamineand 0.35 g of 3-methyl-2-methylthio-benzothiazolium tosylate is heatedin 5 mL of methylene chloride overnight. To the reaction mixture, 10 mLof water is added and filtered and dried in vacuo. The solid is heatedin 10 mL of dichloroethane with 0.25 mL of phosphorous oxychloride for 7hours to obtain Compound 12.

Example 13 Preparation of Compound 13

A mixture of 73 mg of Compound 12 and 76 μL of3,3′-iminobis(N,N-dimethylpropylamine) is heated in 1 mL of DMF at 60 Cfor 2 hours. The reaction mixture is then added to a basic sodium iodidesolution (0.25 g of NaI and 0.5 mL of a 10% NaOH in 10 mL of water). Thesolid is filtered and dried and heated with 1 mL of iodomethane in 1.5mL of DMF at 60 C for 2 hours to obtain Compound 13.

Example 14 Preparation of Compound 14

To 848 mg of 1,4-bis(4-bromobenzyl)piperazine in 60 mL of THF at −78 Cunder nitrogen, 2.5 mL of a 1.6M n-butyllithium is introduced followedby 0.865 g of 1,4-dimethyl-2-quinolone in 20 mL of THF. After one hourof stirring at the low temperature, 2 mL of acetic acid is added andstirred at room temperature overnight. All volatile components areremoved under reduced pressure and the residue is resuspended in 50 mLof methylene chloride and 2.8 g of 3-methyl-2-methylthiobenoxazoliumtosylate and 2 mL of triethylamine is added. The reaction mixture isstirred at room temperature for 15 minutes and Compound 14 is purifiedon a silica gel column.

Example 15 Preparation of Compound 15

A mixture of 0.55 g of2-chloro-4-(2,3-dihydro-3-methyl-(benzo-1,3-thiazol-2-yl)-methylidene-1-phenylquinoliniumchloride, 43 mg of cylcen and 0.2 mL of triethylamine is heated at 60 Cfor three days to obtain Compound 15.

Example 16 Preparation of Compound 16

A mixture of 0.34 g of2-chloro-4-(2,3-dihydro-3-methyl-(benzo-1,3-thiazol-2-yl)-methylidene-1-phenylquinoliniumchloride, 68 mg of 1,4,10,13-tetraoxa-7,16-diaza-cyclooctadecane, and0.11 mL of triethylamine is heated in 10 mL of DMF at 60 C overnight.The crude is precipitated out by the addition of ethyl acetate andpurified on a silica gel column to obtain Compound 16.

Example 17 Preparation of Compound 17

A mixture of 0.6 g of2-chloro-4-(2,3-dihydro-3-methyl-(benzo-1,3-thiazol-2-yl)-methylidene-1-phenylquinoliniumchloride, 18 mg of N,N′-dimethylhydrazine, and 0.35 mL of triethylamineis heated at 80 C for 9 hours. The reaction mixture is then added to 50mL of a 1:1 mixture of brine and water and filtered to obtain the crude,which is then purified by recrystallization from DMF and ethyl acetateto obtain Compound 17.

Example 18 Preparation of Compound 18

A mixture of 0.16 g of2-chloro-4-(2,3-dihydro-3-methyl-(benzo-1,3-thiazol-2-yl)-propylidene-1-phenylpyridiniumchloride, 10 mg of piperazine and 35 μL of triethylamine in 5 mL of DMFat 60 C overnight. The crude is then purified on a silica gel column toobtain Compound 18.

Example 19 Preparation of Compound 19

A mixture of 0.1 g of2-chloro-4-(2,3-dihydro-3-methyl-(benzo-1,3-thiazol-2-yl)-methylidene-7-methyoxy-1-phenylquinoliniumiodide, 5 mg of piperazine, and 25 μL of triethylamine is heated in 5 mLof DMF at 60 C for 2 hours and filtered to obtain Compound 19.

Example 20 Preparation of Compound 20

A mixture of 0.16 g of2-chloro-4-(2,3-dihydro-3-methyl-(benz-1,3-oxazol-2-yl)-methylidene-1-phenylquinoliniumchloride, 7.3 mg of piperazine, and 33 μL of triethylamine is heated in3 mL of DMF at 60 C for 7 hours and filtered to obtain Compound 20.

Example 21 Preparation of Compound 21

A mixture of 0.1 g of2-chloro-4-(2,3-dihydro-3-methyl-(benzo-1,3-thiazol-2-yl)-propylidene-1-phenylpyridiniumchloride, 10 mg of 4-aminopiperidine, and 70 μL of triethylamine isheated in 5 mL of dichloroethane at 60 C overnight to obtain Compound21.

Example 22 Preparation of Compound 22

A mixture of 0.1 g of2-chloro-4-(2,3-dihydro-3-methyl-(benz-1,3-oxazol-2-yl)-methylidene-1-phenylpyridiniumchloride, 25 mg of 1,9-nonanedithiol, and 36 μL of triethylamine isstirred in 5 mL of DMF at room temperature overnight to obtain Compound22.

Example 23 Preparation of Compound 23

A mixture of 0.1 g of2-chloro-4-(2,3-dihydro-3-methyl-(benzo-1,3-thiazol-2-yl)-methylidene-1-phenylpyridiniumchloride, 9 mg of piperazine, and 0.1 mL of triethylamine is heated in 5mL of dichloroethane at reflux overnight. The reaction mixture isfiltered to obtain Compound 23.

Example 24 Preparation of Compound 24

A mixture of2-chloro-4-(2,3-dihydro-3-methyl-(benzo-1,3-thiazol-2-yl)-methylidene-1-phenylquinoliniumchloride, 0.5 equivalent of 1,3-benzenedithiol and 2 equivalents oftriethylamine is stirred at room temperature in methylene chloride toobtain the product.

Example 25 Preparation of Compound 25

A mixture of 118 mg of2-chloro-4-(2,3-dihydro-3-methyl-(benzo-1,3-thiazol-2-yl)-methylidene-1-phenylquinoliniumchloride, 786 mg of 1,4-diaminopiperazine, and 1.1 mL of triethylamineis heated in 10 mL of dichloroethane at 60 C for 24 hours. Theintermediate is recovered from filtration and after purification isheated with 1.2 equivalents of2-chloro-4-(2,3-dihydro-3-methyl-(benzo-1,3-thiazol-2-yl)-methylidene-1-phenylpyridiniumchloride and 3 equivalents of triethylamine in DMF at 60 C for 6 h.Compound 25 is then obtained by filtration.

Example 26 Preparation of Compound 29

A mixture of2-chloro-4-(2,3-dihydro-3-methyl-(benzo-1,3-thiazol-2-yl)-methylidene-1-phenylquinoliniumchloride, 0.5 equivalent of 4-aminomethylpiperidine and 2 equivalents oftriethylamine is heated in DMF at 60 C to yield the product.

Example 27 Preparation of Compound 30

A mixture of2-chloro-4-(2,3-dihydro-3-methyl-(benzo-1,3-thiazol-2-yl)-methylidene-1-phenylquinoliniumchloride, 0.5 equivalent of 3-aminopyrrolidine dihydrochloride and 4equivalent of triethylamine is heated in DMF at 60 C to yield theproduct.

Example 28 Preparation of Compound 31

A mixture of 3-(5-carboxypentyl)-2-methylthio-benzothiazolium bromideand one equivalent of 2-chloro-4-methyl-1-phenylquinolinium chloride andtwo equivalents of triethylamine is stirred in DMF at room temperatureto yield the product.

Example 29 Preparation of Compound 32

A mixture of2-chloro-4-(2,3-dihydro-3-(5-carboxypentyl)-(benzo-1,3-thiazol-2-yl)-methylidene-1-phenylquinoliniumchloride (Compound 31), 0.5 equivalent of piperazine and 6 equivalentsof triethylamine is heated in DMF at 60 C and the diacid isolated isconverted to the corresponding bis succinimidyl ester withO—(N-succinimidyl)-N,N,N′,N′-tetramethyluronium tetrifluoroborate andtriethylamine in DMF.

Example 30 Preparation of Compound 33

The compound is prepared by treating Compound 32 with 2 equivalent ofN-2(aminoethyl)maleimide trifluoroacetic acid salt in the presence of 5equivalents of triethylamine in DMF at room temperature.

Example 31 Preparation of Compound 34

The compound is prepared by treating Compound 32 with 10 equivalents ofhydrazine at room temperature in DMF.

Example 32 Preparation of Compound 35

A mixture of2-chloro-4-(2,3-dihydro-3-methyl-(benzo-1,3-thiazol-2-yl)-methylidene-1-phenylquinoliniumchloride, 0.5 equivalent of 4-amino-4H-1,2,4-triazole-3,5-dithiol and 3equivalent of triethylamine is stirred in methylene chloride at roomtemperature to yield the product.

Example 33 Preparation of Compound 36

A mixture of Compound 35 and 1.5 equivalent of succinic anhydride isheated in DMF at about 50 C to generate the target carboxylic acid whichis converted to the corresponding succinimidyl ester withO—(N-succinimidyl)-N,N,N′,N′-tetramethyluronium tetrifluoroborate andtriethylamine in DMF.

Example 34 Preparation of Compound 37

The compound is prepared by treating Compound 36 with 2 equivalent ofN-2(aminoethyl)maleimide trifluoroacetic acid salt in the presence of 5equivalents of triethylamine in DMF at room temperature.

Example 35 Emission Spectra of Compound 6 and Compound 19 Binding to RNAand DNA Demonstrating a Fluorescent Signal Intensity that is Greater forRNA than DNA

A stock solution of Compound 6 and Compound 19 was made by dissolving anunknown mass of each reporter molecule individually in 1 mL of DMSO. Thestock solution was then diluted 1/1261 in 10 mM TRIS, 1 mM EDTA (pH7.2). This dilute solution resulted in an OD at ˜0.05-0.1 and extinctioncoefficient set at 45,000 yielding a working concentration of ˜1-2 μM.Compound 6 and Compound 19, each at about 1-2 μM, were added to the testsamples (1) Buffer only; 2) DNA calf thymus; 3) DNA Type XI, Micrococcuslysodeikticus; 4) DNA Type XII Clostridium perfringens; 5) DNA TypeVIII, E. coli strain B; 6) RNA, ribosomal; 7) RNA Type III, Baker'syeast and 8) RNA Type XX, E. coli strain W). The RNA and DNA werepresent at a final concentration of about 60 μg/mL. After addition ofthe dye and the nucleic acid the samples were excited at 470 nm andemission read. See, FIG. 1.

This example provides a methodology for testing compounds within thescope of the invention for their ability to fluoresce when complexedwith RNA or DNA. In addition, this methodology provides a method forscreening compounds wherein a particular intensity is desired orcompounds that are selective for RNA and/or DNA, showing a greatersignal intensity for one form of nucleic acid in the presence of anotherform.

Example 36 Comparison of in Solution Binding of Compound 11 to RNA, DNAand RNA+DNA

A buffer solution of 200 ul of 10 mM TRIS, 1 mM EDTA (pH7.2) was addedto the wells of a 96-well microplate. RNA and DNA (calf thymus,Micrococcus lysodeikticus and Clostridium perfringens) dilutions in TE(pH7.2) were added to the appropriate wells to yield the finalconcentrations of 0-150 ng/mL. In separate wells RNA and DNA werecombined for a final concentration of 150 ng/mL of nucleic acid whereinthe concentrations were combined in the following format: RNA+DNArespectively, 0 ng/mL+150 ng/mL, 25 ng/mL+125 ng/mL, 50 ng/mL+100 ng/mL,75 ng/mL+75 ng/mL, 100 ng/mL+50 ng/mL, 125 ng/mL+25 ng/mL and 150ng/mL+0 ng/mL. Compound 11, from a stock solution in DMSO, was added tothe microplate wells at a final concentration of 0.015 μM. The wellswere read at 460 nm/500 nm Ex/Em. Compound 11 demonstrated an increasedfluorescent intensity signal with increasing concentrations of RNA butlittle to no signal when combined with DNA alone. These results wereconfirmed wherein the combined RNA+DNA resulted in the same signalintensity for the corresponding concentration of RNA. See, FIG. 2.

Using this or similar methodology, a variety of compounds as describedherein may be screened for their fluorescence properties when associatedwith nucleic acids. Similarly, compounds may be screened based upontheir relative fluorescence enhancement when associated with RNA versusDNA, or alternatively for their relative fluorescence enhancement whenassociated with DNA in the presence of RNA. Compounds can be readilyscreened for a particular desired intensity, wavelength, or selectivityfor RNA and/or DNA. See, Table 3

TABLE 3 Compound Excitation/Emission (nm)¹ Fluorescence EnhancementRatio (RNA/DNA)²  1 M 1.0  6 M 7.0  7 M 2.3  8 M 4.9  9 M 1.7 11 M 12 13M 2.5 18 570/620 1.3 19 M 4.9 20 M 8.5 25 M 5.6 Fluorescence EnhancementRatio (DNA/RNA)³ 15 M 3.7 ¹Complex with nucleic acid ²The ratio of thefluorescence enhancement of the compound when associated with RNA to thefluorescence enhancement of the compound when associated with DNA. ³Theratio of the fluorescence enhancement of the compound when associatedwith DNA to the fluorescence enhancement of the compound when associatedwith RNA. M = excitation of about 480-510 nm and emission of about510-540 nm.

Example 37 Rifampicin-Depletion of Bacterial RNA

Gram-positive (Staphylococcus aureus) and Gram-negative (Eshcerichiacoli) bacteria in growth medium were treated with 25 μg/mL rifampicinfor one hour. Treated and untreated bacteria were counted, diluted to1×10⁷ bacteria/mL in 0.85% NaCl, and 1 mL aliquots incubated with 0.25μM, 0.5 μM, or 1 μM Compound 6 or Compound 9 for 10-15 minutes protectedfrom light. The samples were analyzed by flow cytometry and then 100 μLvolumes were transferred to a microplate in triplicate and analyzed by amicroplate reader. The fluorescence intensity was corrected against thedye in solution, which was weakly fluorescent. Results demonstrate adecrease in fluorescent signal after removal of mRNA in comparison tountreated cells, See FIG. 3.

Example 38 Nucleic Acid Binding of Compound 6 after RNase and DNase onMethanol-Fixed Eukaryotic Cells

Bovine pulmonary arterial endothelial cells were grown in media for oneday after plating and then fixed ten minutes in 100% methanol at −20° C.Cells were washed 3×5 minutes in PBT (0.05M phosphate-buffered saline(PBS) containing 0.1% Triton X-100) and then treated with RNase (fromRoche, at 1:500 dilution) or DNase (from Promega, at 1:100 dilution) for2 hours at 37° C. Cells were washed again for 3×5 minutes in PBT andthen labeled with Compound 6 at 687 nM concentration for 20 minutes.Cells were washed 3×10 minutes in PBS and mounted on microscope slidesin ProLong antifade mounting medium. After the mountant had hardened,slides were examined on a Nikon Eclipse 800 upright fluorescentmicroscope and imaged with a FITC filter set, a Princeton InstrumentMicroMax cooled CCD camera, and Universal Imaging MetaMorph imagingsoftware. Both by eye and through intensity quantification, resultsshowed that RNase washing eliminated Compound 6 labeling of nucleoli(where RNA is most strongly localized). DNase did not significantlyaffect nucleolar intensity. Nuclear labeling was significantly reducedby RNase, but not DNase. By eye, cytoplasmic label was reduced by RNasebut not DNase. To test the activity of the DNase, a DAPI DNA labelcontrol was treated with RNase and showed a significant decline in labelintensity. See, FIG. 4.

Example 39 Nucleic Acid Binding of Compound 6 and Compound 19 on RNaseand DNase Treated Formaldehyde-Fixed Eukaryotic Cells

Bovine pulmonary arterial endothelial cells were grown in media for oneday after plating and then fixed fifteen minutes in 4% paraformaldehyde.Cells were washed 3×5 minutes in PBT (0.05M phosphate-buffered saline(PBS) containing 0.1% Triton X-100) and then treated with RNase (fromRoche, at 1:500 dilution) or DNase (from Promega, at 1:100 dilution), orboth for 2 hours at 37° C. Cells were washed again for 3×5 minutes inPBT and then labeled with Compound 6 at 687 nM concentration or Compound19 at 1 μM concentration in PBS for 20 minutes. Cells were washed 3×10minutes in PBS and mounted on microscope slides in ProLong antifademounting medium (Molecular Probes, Inc.). After the mountant hadhardened, slides were examined on a Nikon Eclipse 800 uprightfluorescent microscope and imaged with a FITC filter set, a PrincetonInstrument MicroMax cooled CCD camera, and Universal Imaging MetaMorphimaging software. Both by eye and through intensity quantification,results showed that RNase washing eliminated Compound 6 labeling ofnucleoli (where RNA is most strongly localized). The same was true forCompound 19. DNase significantly reduced Compound 6 and Compound 19nucleolar intensity. Nuclear labeling with Compound 6 was significantlyreduced by both RNase and DNase. Nuclear labeling with Compound 19 wassignificantly reduced with RNase, but not DNase. For Compound 6,cytoplasmic label (which is more pronounced withparaformaldehyde-fixation than with methanol fixation) was significantlyreduced by both RNase and DNase. With Compound 19, cytoplasmic label wasnot reduced at all with either RNase or DNase washing. See, FIG. 5.

Example 40 Nucleic Acid Binding of Compound 6 in Live Eukaryotic CellsFollowed by Fixing of Cells

Bovine pulmonary arterial endothelial cells were grown in media for oneday after plating and then labeled before fixing with 687 nM Compound 6for 20 minutes. Some cells were rinsed in media and separated for liveimaging. The other cells were rinsed twice with media and then fixed in4% paraformaldehyde for 15 minutes at room temperature, 0.5%glutaraldehyde for 5 minutes at room temperature, or 100% methanol for10 minutes at −20° C. Cells were washed 3×10 minutes inphosphate-buffered saline and mounted on microscope slides in ProLongantifade mounting medium (Molecular Probes, Inc.). After the mountanthad hardened, slides were examined on a Nikon Eclipse 800 uprightfluorescent microscope and imaged with a FITC filter set, a PrincetonInstrument MicroMax cooled CCD camera, and Universal Imaging MetaMorphimaging software. Live cells showed strong nucleolar label, strongpunctate labeling in mitochondria consistent with mtDNA, and dimmerubiquitous mitochondrial labeling. Fixation with either paraformaldehydeor glutaraldehyde removed all mitochondrial labeling. Nucleolar labelremained, but nuclear label increased. General cytoplasmic labeling alsoincreased significantly. Methanol-fixation had good nucleolar labeling,with only a slight increase in nuclear labeling. All mitochondriallabeling was removed. Cytoplasmic label increased slightly, but notnearly to the extent seen with other fixation. Results generallyindicate that methanol-fixation is the recommended fixation technique.

Example 41 Nucleic Acid Binding of Compound 21 in Live Eukaryotic Cells

Bovine pulmonary arterial endothelial (BPEA) cells and HeLa humancervical cancer cells were grown in media for one day after plating oncoverslips. Cells were labeled with 1 μM or 10 μM of Compound 21 forHeLa cells, or 10 μM in BPAE cells, in media at 37° C. for 20 minutes,then rinsed twice and mounted in warm medium. Coverslips were examinedon a Nikon Eclipse 800 upright fluorescent microscope and imaged with afar red filter set, a Princeton Instruments MicroMax cooled CCD camera,and Universal Imaging MetaMorph imaging software. Compound 20 was cellpermeable in live cells and in HeLa cells showed mitochondrial labeling,with stronger punctate spots within the mitochondria, typical ofmitochondrial DNA staining.

Also seen was very dim nucleolar labeling. The labeling in the cells wasthe same for the 1 μM or 10 μM concentration of Compound 20. In BPAEcells, there was no specific labeling evident, and much higher off-cellbackground. While Compound 20 demonstrated a fluorescence enhancementratio (RNA/DNA) of 8.5 (See, Table 3), Compound 20 did not demonstrateselective detection of RNA in live cells.

Example 42 Nucleic Acid Binding of Compound 6 and Compound 11Methanol-Fixed Eukaryotic Cells

Bovine pulmonary arterial endothelial cells were grown in media for oneday after plating on coverslips and then fixed 10 minutes in −20° C.100% methanol. Cells were washed 3×5 minutes 50 mM phosphate bufferedsaline (PBS), pH 7.3. Cells were labeled with Compound 11 at 5 μM, 1 μM,or 500 nM concentration in PBS for 20 minutes, washed 3×10 minutes inPBS, and mounted in ProLong antifade mountant (Molecular Probes, Inc.).Cells were labeled with Compound 6 at 500 nM in PBS for 20 minutes.After the mountant had hardened, slides were examined on a Nikon Eclipse800 upright fluorescent microscope and imaged with a blue-green filterset, a Princeton Instruments MicroMax cooled CCD camera, and UniversalImaging MetaMorph imaging software. Comparison of emission wavelengthwas made using a Zeiss 510 confocal microscope utilizing a META spectralunmixing system. The resultant staining pattern for both Compound 11 andCompound 6 showed strong nucleolar and cytoplasmic labeling, with dimmernuclear labeling, indicating selective RNA staining. Optimal labelingfor both compounds appeared to be about 500 μM. Spectral comparison ofthe emission wavelengths for Compound 11 showed a peak emission around500 nm, while Compound 6 peaked around 530 nm. See, FIG. 6.

Example 43 Demonstration of Cell Permeability and Basic Nucleic AcidLabel Pattern with a Concentration Series of Compound 1 in Live Cells

Bovine pulmonary arterial endothelial cells were grown in media for oneday after plating on coverslips, then labeled with 50 nM MitoTracker RedCMXRos (Molecular Probes, Inc., M7512) in Hank's balanced salt solution(HBSS) with HEPES for general mitochondrial labeling. After washing inHBSS, cells were then labeled with Compound 1 at various concentrationsranging from 100 nM up to 10 μM in media for 20 minutes at 37°. Cellswere washed well with HBSS. Coverslips were examined mounted in warmHBSS on a Nikon Eclipse 800 upright fluorescent microscope and imagedwith a FITC filter set, a Princeton Instruments MicroMax cooled CCDcamera, and Universal Imaging MetaMorph imaging software. At 100 nM to500 nM, the resultant staining pattern showed what appeared to bemitochondrial DNA (mtDNA), with no nuclear label, or cytoplasmicbackground. The mtDNA signal was localized within mitochondria, asevidenced by MitoTracker Red labeling. When the concentration ofCompound 1 was increased to 1 μM, nuclear labeling was seen as well asmtDNA. At 3 μM of Compound 1 or higher, mitochondrial morphology wasdisrupted, as evidenced with MitoTracker Red, and mtDNA labeling waslost, replaced with ubiquitous cytoplasmic labeling and strong nuclearlabeling.

The preceding examples can be repeated with similar success bysubstituting the specifically described nucleic acid reporter moleculesof the preceding examples with those generically and specificallydescribed in the forgoing description. One skilled in the art can easilyascertain the essential characteristics of the present invention, andwithout departing from the scope thereof, can make various changes andmodifications of the invention to adapt to various usages andconditions.

All publications, patents and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent or patent application wasspecifically and individually indicated to be incorporated by reference.

1. A complex comprising: a) a nucleic acid reporter molecule comprisinga first nucleic acid complexing monomer moiety, a second nucleic acidcomplexing monomer moiety and a linker that comprises at least onearomatic, heteroaromatic, cyclic or heterocyclic moiety comprising 3-20non-hydrogen atoms selected from the group consisting of O, N, S, P andC, wherein the first nucleic acid complexing monomer moiety iscovalently attached to the linker; and the second nucleic acidcomplexing monomer moiety is covalently attached to the linker; and, b)a nucleic acid molecule.
 2. The complex according to claim 1, whereinthe first and the second first nucleic acid complexing monomer moietiesare the same.
 3. The complex according to claim 1, wherein the first andthe second first nucleic acid complexing monomer moieties are different.4. A complex comprising: a) a nucleic acid reporter molecule accordingto the formula

wherein each R³ is independently hydrogen, unsubstituted C₁-C₆ alkyl,substituted C₁-C₆ alkyl, unsubstituted C₁-C₆ alkoxy, substituted C₁-C₆alkoxy, unsubstituted fused benzene, substituted fused benzene,unsubstituted trifluoromethyl, substituted trifluoromethyl,unsubstituted halogen, substituted halogen, unsubstituted reactivegroup, substituted reactive group, unsubstituted carrier molecule,substituted carrier molecule, unsubstituted solid support or substitutedsolid support; each R⁴ is independently a unsubstituted C₁-C₆ alkyl,substituted C₁-C₆ alkyl, unsubstituted reactive group, substitutedreactive group, unsubstituted carrier molecule, substituted carriermolecule, unsubstituted solid support or substituted solid support; X isO, S, or CR⁶R⁷ wherein each R⁶ and R⁷ are independently a unsubstitutedC₁-C₆ alkyl, substituted C₁-C₆ alkyl, unsubstituted reactive group,substituted reactive group, unsubstituted carrier molecule, substitutedcarrier molecule, unsubstituted solid support, substituted solid supportor R⁶ and R⁷ taken together form a 5- or 6-membered saturated ring; j is0, 1, or 2; linker is a series of stable covalent bonds comprising 1-30nonhydrogen atoms selected from the group consisting of C, N, O, S andP; and, K is substituted pyridinium, unsubstituted pyridinium,substituted quinolinium, or unsubstituted quinolinium; and, b) a nucleicacid molecule.
 5. A complex according to claim 4, wherein the nucleicacid molecule is single stranded RNA, double stranded RNA, singlestranded DNA or double stranded DNA.
 6. The complex according to claim4, wherein the molecule is


7. The complex according to claim 4, wherein the linker comprises atleast one aromatic, heteroaromatic, cyclic or heterocyclic moietycomprising 3-20 non-hydrogen atoms selected from the group consisting ofO, N, S, P and C.
 8. The complex according to claim 7, wherein thelinker has the formula (I)—(Y)_(r)—(CH₂)_(m)-T_(q)-(CH₂)_(n)-E-(CH₂)_(n)-T_(q)-(CH₂)_(m)—(Y)_(r)—;wherein Y is a linear or branched moiety comprising 1-20 non-hydrogenatoms selected from the group consisting of C, N, O, P and S; T is aunsubstituted heteroatom or a substituted heteroatom; E is an aromatic,heteroaromatic, cyclic or heterocyclic moiety comprising 3-20non-hydrogen atoms selected from the group consisting of O, N, S, P andC; r is independently 0 or 1; m is an integer of 0-6; n is independently0 or 1; q is independently 0 or 1; and wherein the first unsymmetricalcyanine monomer moiety is covalently attached to the linker; and thesecond unsymmetrical cyanine monomer moiety is covalently attached tothe linker.
 9. The complex according to claim 8, wherein E is

wherein R² is hydrogen, amine, substituted amine, substituted C₁-C₆alkyl, unsubstituted C₁-C₆ alkyl, unsubstituted reactive group,substituted reactive group, unsubstituted carrier molecule, substitutedcarrier molecule, unsubstituted solid support, or substituted solidsupport.
 10. The complex according to claim 9, wherein T, when present,is —NR², —N═ or S wherein the R² is hydrogen, amine, substituted amine,substituted C₁-C₆ alkyl, unsubstituted C₁-C₆ alkyl, unsubstitutedreactive group, substituted reactive group, unsubstituted carriermolecule, substituted carrier molecule, unsubstituted solid support, orsubstituted solid support.
 11. The complex according to claim 10,wherein the reactive group, solid support and carrier molecule comprisea linker that is a single covalent bond, or a covalent linkage that islinear or branched, cyclic or heterocyclic, saturated or unsaturated,having 1-20 nonhydrogen atoms selected from the group consisting of C,N, P, O and S; and are composed of any combination of ether, thioether,amine, ester, carboxamide, sulfonamide, hydrazide bonds and aromatic orheteroaromatic bonds.
 12. The complex according to claim 11, wherein thereactive group is selected from the group consisting of an acrylamide,an activated ester of a carboxylic acid, a carboxylic ester, an acylazide, an acyl nitrile, an aldehyde, an alkyl halide, an anhydride, ananiline, an amine, an aryl halide, an azide, an aziridine, a boronate, adiazoalkane, a haloacetamide, a haloalkyl, a halotriazine, a hydrazine,an imido ester, an isocyanate, an isothiocyanate, a maleimide, aphosphoramidite, a reactive platinum complex, a silyl halide, a sulfonylhalide, a thiol and a photoactivatable group.
 13. The complex accordingto claim 12, wherein the reactive group is selected from the groupconsisting of carboxylic acid, succinimidyl ester of a carboxylic acid,hydrazide, amine and a maleimide.
 14. The complex according to claim 11,wherein the carrier molecule is selected from the group consisting of anamino acid, a peptide, a protein, a polysaccharide, a nucleoside, anucleotide, an oligonucleotide, a nucleic acid polymer, a hapten, apsoralen, a drug, a hormone, a lipid, a lipid assembly, a syntheticpolymer, a polymeric microparticle, a biological cell or a virus. 15.The complex according to claim 14, wherein the carrier molecule isselected from the group consisting of an antibody or fragment thereof,an avidin or streptavidin, a biotin, a blood component protein, adextran, an enzyme, an enzyme inhibitor, a hormone, an IgG bindingprotein, a fluorescent protein, a growth factor, a lectin, alipopolysaccharide, a microorganism, a metal binding protein, a metalchelating moiety, a non-biological microparticle, a peptide toxin, aphosphotidylserine-binding protein, a structural protein, asmall-molecule drug, or a tyramide.
 16. The complex according to claim11, wherein the solid support is selected from the group consisting of amicrofluidic chip, a silicon chip, a microscope slide, a microplatewell, silica gels, polymeric membranes, particles, derivatized plasticfilms, glass beads, cotton, plastic beads, alumina gels,polysaccharides, polyvinylchloride, polypropylene, polyethylene, nylon,latex bead, magnetic bead, paramagnetic bead, and superparamagneticbead.
 17. The complex according to claim 16, wherein the solid supportis selected from the group consisting of Sepharose, poly(acrylate),polystyrene, poly(acrylamide), polyol, agarose, agar, cellulose,dextran, starch, FICOLL, heparin, glycogen, amylopectin, mannan, inulin,nitrocellulose, diazocellulose and starch.
 18. The complex according toclaim 9, wherein the linker is

each R⁸, R⁹, R¹⁰, R¹¹ and R¹² are independently a —NR², —N═ or S whereinR² is hydrogen, amine, substituted amine, substituted C₁-C₆ alkyl,unsubstituted C₁-C₆ alkyl, unsubstituted reactive group, substitutedreactive group, unsubstituted carrier molecule, substituted carriermolecule, unsubstituted solid support, or substituted solid support;each Z is independently —(CH₂)_(g)— wherein g is 0 or 1, wherein thedashed line is attached directly to the K moiety of the unsymmetricalcyanine monomer.
 19. The complex according to claim 18, wherein R⁸ is S.20. The complex according to claim 18, wherein R¹² is —NH or —N═. 21.The complex according to claim 18, wherein the linker is